Differentiation marker and differentiation control of eye cell

ABSTRACT

The present invention relates to a differentiation marker and a differentiation controlling technique for an eye cell. More particularly, the present invention has attained an object of providing a differentiation marker for an eye cell among the aforementioned problems, by providing a marker for identifying a cell having a high proliferation ability among corneal endothelial cells and/or the differentiation ability of a corneal endothelial cell, the marker comprising GPR49/LGR5, as well as a detection agent or detection method for identifying a cell having a high proliferation ability among corneal endothelial cells and/or the differentiation ability of a corneal endothelial cell, comprising a substance binding to GPR49/LGR5. In addition, the present invention has attained an object of providing a differentiation controlling technique for an eye cell, by providing an agent for suppressing differentiation and/or promoting proliferation of an eye cell, comprising R-spondins.

TECHNICAL FIELD

The present invention relates to a cell, particularly, a marker in adifferentiated state in the ophthalmological region, a technique and amethod for suppressing differentiation and/or stimulating proliferationof an eye cell (particularly, corneal endothelial cell which isdifficult in differentiation control), as well as an agent and a culturemedium therefore.

BACKGROUND ART

Visual information is recognized in such a manner that light transmittedinto the cornea, which is a transparent tissue at the forefront of aneyeball, reaches the retina to excite the nerve cell of the retina, andan electric signal generated is transferred through the optic nerve tothe visual cortex of the cerebrum. In order to obtain good visualacuity, it is necessary that the cornea is transparent. The transparencyof the cornea is maintained by retaining a constant moisture contentwith the pumping function and barrier function of a corneal endothelialcell.

The cornea is a transparent tissue which is positioned in front of theeyeball and the structure of cornea is mainly composed of three-layersknown as corneal epithelial cell layer, corneal stromal layer andcorneal endothelial cell layer. The corneal endothelial cell layer is asingle cell layer existing in a corneal deep part, has the barrierfunction and the pumping function, and plays a role in maintaining thetransparency of the cornea by retaining a constant corneal moistureamount. In addition, it is known that even if the corneal endothelialcells are damaged, they do not proliferate in a living body, and also itis known that a serious visual disorder is generated by damaging cornealendothelial cells with trauma, a disease or the like to decrease theirnumber.

Human corneal endothelial cells exist at a density of about 3000 cellsper 1 square millimeter at birth, but once the corneal endothelial cellsare damaged, they have no ability to regenerate themselves. Inendothelial corneal dystrophy or bullous keratopathy which is generatedby dysfunction of the corneal endothelium due to a variety of causes,the cornea becomes opaque due to edema, which leads to remarkablereduction in the visible acuity. Currently, penetrating keratoplasty fortransplanting the whole three-layered structure of epithelium, stromaand endothelium of the cornea is performed for bullous keratopathy.However, donation of the cornea in Japan is insufficient, and the numberof waiting patients for corneal transplantation is about 2600, but thenumber of corneal transplantation performed by using the cornea from adonor in Japan per year is about 1700.

In recent years, for the purpose of reducing the risk of immunologicalrejection or the risk of postoperative complications and obtainingbetter visual function, the idea of “parts transplantation” fortransplanting a damaged tissue alone has been drawing the attention.Among the cornea transplantations, deep layer and superficial layercorneal transplantations in which the stromal tissue is transplanted,Descemet's Stripping Automated Endothelial Keratoplasty in which thecorneal endothelial tissue is transplanted, and the like have beenperformed. In addition, cultured mucosal epithelial transplantation inwhich the corneal epithelium or the oral mucosa which has been culturedin vitro is transplanted in place of the corneal epithelium has beenalready applied clinically, and a method for transplanting the cornealendothelium which has been cultured in vitro similarly has been alsostudied.

It is known that a corneal endothelial cell is differentiated whencultured, and morphology changes to a fibroblast cell-like form. Inaddition, it is known that GPR49/LGR5 is expressed in a small intestineepithelial stem cell in a limited manner, and plays an important role.As a ligand of GPR49/LGR5, R-spondins are reported (Non-Patent Documents1 to 4).

Non-Patent Document 5 describes that a stem cell-like cell exists in acorneal limbal epithelial cell, and GPR49/LGR5 can be a phenotype markerof a remaining human corneal limbal epithelial stem cell.

In Non-Patent Document 6, GPR49/LGR5 is exemplified as a stem cellmarker. Non-Patent Document 6 describes that, in two cell populations ofmouse corneal epithelial cells, GPR49/LGR5 and ABCG2 are highlyexpressed.

Non-Patent Document 7 describes GPR49/LGR5 as a stem cell marker.

Non-Patent Document 8 discloses that intestinal tract epithelial stemcell culturing is established.

Non-Patent Document 9 discloses a method of small intestinal culture anda large intestinal culture in a stable manner and for a long term.Non-Patent Document 9 describes that culture growth is markedlystimulated by a fusion protein (RSpo1-Fc) between R-spondin 1 andimmunoglobulin Fc.

Previously, it has been considered that corneal endothelial cells do notproliferate in vivo, but by molecular biological or cellular biologicalstudy in recent years, it has been reported that a cell group very richin proliferation ability also exists in the corneal endothelial celllayer. Schimmelpfenning et al. revealed that the endothelial celldensity is increased more at a peripheral part of the human cornea thanat a central part, and proposed a possibility that the proliferation ofcells at a corneal peripheral part supplies cells to a corneal centralpart (Non-Patent Document 10).

In addition, using the rabbit cornea, Whikehart et al. confirmed thattelomerase, which is observed to be highly expressed in a stem cell or aprecursor cell, is highly expressed in an endothelial cell at a cornealperipheral part. Further, by an evaluation method of using BrdU as acell proliferation marker, it is shown that a fast response is observedwhen an endothelial cell at a corneal peripheral part is damaged(Non-Patent Document 11). In addition, Yokoo et al. succeeded incollection of a precursor cell of a corneal endothelial cell from theadult human cornea using a sphere method which is a method forcollecting a mesenchymal stem cell (Non-Patent Document 12). This cellexpresses an undifferentiated cell marker, and when the ability to forma sphere was compared between corneal endothelial cells at a centralpart and a peripheral part, it was confirmed that the ability is high ina peripheral endothelial cell (Non-Patent Document 13). Furthermore,McGowan et al. reported that a large number of cells expressing anundifferentiated cell marker exist at a corneal peripheral part, andthese cells are activated by being damaged (Non-Patent Document 14).

G protein-coupled receptor 49 (also referred to as “GPR49/LGR5” as usedherein) is one kind of seven-transmembrane receptors similar tothyroid-stimulating hormone, follicle-stimulating hormone (FSH), orleuteinizing hormone (LH), and has a unique structure associated with anextracellular N-terminal domain including a leucine rich repeat (FIG. 1,Non-Patent Document 15). It has been reported that since GPR49/LGR5 is atarget gene of Wnt-signaling pathway and Hedgehog-signaling pathwayinvolved in oncogenesis, expression of GPR49/LGR5 is elevated byabnormality of these signaling pathways (Non-Patent Document 16,Non-Patent Document 17 and Non-Patent Document 18). Furthermore, sinceit was confirmed that specific expression of GPR49/LGR5 is seen in anintestinal tract epithelial stem cell (Non-Patent Document 8),GPR49/LGR5 has been drawing attention as a novel protein which isexpressed in a stem cell-specific manner. Thereafter, it has beenconfirmed that expression of GPR49/LGR5 is elevated in a stemcell-specific manner also in a tissue such as hair follicle (Non-PatentDocument 19) or stomach epithelium (Non-Patent Document 20), and apossibility that GPR49/LGR5 is involved in construction of stem cellniche and tissue formation has been also reported.

PRIOR ART DOCUMENT Patent Documents

Non-Patent Document 1: Carmon K S. et al., Proc Natl Acad Sci USA. 2011Jul. 12; 108(28) : 11452-11457

Non-Patent Document 2: de Lau W. et al., Nature. 2011 Jul. 4; 476(7360):293-297

Non-Patent Document 3: Glinka A. et al., EMBO Rep. 2011 Sep. 30; 12(10): 1055-1061

Non-Patent Document 4: J. Yoon, J. Lee, Cellular Signalling 24 (2012)369-377

Non-Patent Document 5: Brzeszczynska J, et al., Int J Mol Med. 2012 May;29(5) :871-876

Non-Patent Document 6: Krulova M, et al., Invest Ophthalmol Vis Sci.2008 September; 49(9) : 3903-3908

Non-Patent Document 7: HOLAN V., Ophthalmologica Volume 88, IssueSupplements 246, page 0, September 2010

Non-Patent Document 8: Barker N., et al., Nature 449: 1003-1007, 2007

Non-Patent Document 9: Ootani A., Li X. et al., Nat Med. 2009 June;15(6) : 701-706

Non-Patent Document 10: Schimmelpfennig B., et al., IOVS, Vol. 25, pp.223-229. 1984

Non-Patent Document 11: Whikehart D R., et al., Mol. Vis., 11, pp.816-824, 2005

Non-Patent Document 12: Yokoo S. et al, IOVS. 46, 1626-1631, 2005

Non-Patent Document 13: Yamagami S., et. al., Ophthalmology, 114,433-439, 2007

Non-Patent Document 14: McGowan S L., et al., Mol. Vis., 13:1984-2000.2007

Non-Patent Document 15: Hsu S Y., et al., J. Mol. Endocrinol. 12,1830-1845, 1998

Non-Patent Document 16: Yamamoto Y., et al., Hepatology. 37, 528-533,2003

Non-Patent Document 17: McClanahan T., et al., Cancer Biol Ther.5,419-426, 2006

Non-Patent Document 18: Tanese K., et al., Am J Pathol. 173, 835-843,2008

Non-Patent Document 19: Jaks V., et al., Nature Genetics. 40, 1291-1299,2008

Non-Patent Document 20: Barker N., et al., Cell Stem Cell. 7, 656-670,2010

SUMMARY OF THE INVENTION Solutions to the Problems

The present invention provides a technique for using GPR49/LGR5 as amarker of proliferation/differentiation. The present inventors havefound that GPR49/LGR5 is strongly expressed in a cornea endothelial cell(particularly, peripheral part) in a human corneal tissue; found thatthe expression amount of GPR49/LGR5 is significantly reduced in acultured cell of a human cornea endothelial cell; and also found that aGPR49/LGR5-positive cell group has a small cell size, and has a highproliferation ability. Based on these findings, the present inventorshave applied the above findings to a technique for using GPR49/LGR5 as amarker of proliferation/differentiation.

The present invention also provides use of R-spondins as an agent forsuppressing differentiation or promoting proliferation.

The present inventors have found a tendency such that thedifferentiation of a human cultured corneal endothelial cell issuppressed, and the proliferation of the cell is promoted by R-spondins,particularly R-spondin 1. The present inventors have applied the abovefinding to a technique for using R-spondins as applications such as aliquid for corneal preservation, a liquid for culturing cornealendothelial cell, a therapeutic agent for corneal endothelial celldisorder (eye drops, cell infusion) and an agent for preventingprogression of corneal endothelial cell disorder.

In the previous studies, there is no report on clear presentation of thepresence of a corneal endothelial stem cell. In addition, theproliferation ability of a corneal endothelial cell and the mechanism ofcontrolling undifferentiation in a living body have not been elucidated.The present inventors have found that there is GPR49/LGR5 as a proteinwhich is specifically expressed in a precursor cell of a cornealendothelial cell, and have verified the in vivo and in vitro functionalrole, resulting in an application as a marker.

In one aspect, the present invention provides an agent for suppressingdifferentiation and/or promoting proliferation of a cell, comprising atleast one kind selected from the group consisting of R-spondins and afunctional equivalent thereof.

In one embodiment, the present invention provides an agent forsuppressing differentiation and/or promoting proliferation of a cellselected from an eye cell, a nerve cell including a cell derived from aneural crest cell (including corneal endothelial cell), and epithelialcells such as a conjunctival epithelial cell, an amniotic epithelialcell, an oral mucosal epithelial cell, a nasal mucosal epithelial cell,and a corneal epithelial cell, comprising at least one kind selectedfrom the group consisting of R-spondins and a functional equivalentthereof.

In another embodiment, the present invention provides an agent forsuppressing differentiation and/or promoting proliferation of an eyecell, comprising at least one kind selected from the group consisting ofR-spondins and a functional equivalent thereof.

In another embodiment, the R-spondins in the present invention includeat least one selected from R-spondin 1, R-spondin 2, R-spondin 3 andR-spondin 4.

In a specific embodiment, the R-spondins include R-spondin 1.

In another embodiment, the eye cell is a cell which does not proliferatein the stationary state.

In a specific embodiment, the eye cell includes at least one kind cellselected from a retinal cell, a vitreous body cell, a corneal epithelialcell, a corneal parenchymal cell and a corneal endothelial cell.

In a specific embodiment, the eye cell includes a corneal endothelialcell.

In a specific embodiment, the eye cell includes a corneal endothelialcell of a primate.

In a specific embodiment, the eye cell includes a human cornealendothelial cell.

In a specific embodiment, the eye cell is in the confluent state.

In one aspect, the present invention provides an agent for suppressingdifferentiation and/or promoting proliferation of a cell, comprising atleast one kind selected from the group consisting of SHH, an agonist ofSHH (e.g., agonist of Frizzled family such as purmorphamine) and afunctional equivalent thereof.

In one embodiment, the present invention provides an agent forsuppressing differentiation and/or promoting proliferation of a cellselected from an eye cell, a nerve cell including a cell derived from anneural crest cell (including corneal endothelial cell), and epithelialcells such as a conjunctival epithelial cell, an amniotic epithelialcell, an oral mucosal epithelial cell, a nasal mucosal epithelial cell,and a corneal epithelial cell, comprising at least one kind selectedfrom the group consisting of SHH, purmorphamine and a functionalequivalent thereof.

In another embodiment, the present invention provides an agent forsuppressing differentiation and/or promoting proliferation of an eyecell, comprising at least one kind selected from the group consisting ofSHH, an agonist of SHH (e.g., agonist of Frizzled family such aspurmorphamine) and a functional equivalent thereof.

In another embodiment, the eye cell is a cell which does not proliferatein the stationary state.

In a specific embodiment, the eye cell includes at least one kind cellselected from a retinal cell, a vitreous body cell, a corneal epithelialcell, a corneal parenchymal cell and a corneal endothelial cell.

In a specific embodiment, the eye cell includes a corneal endothelialcell.

In a specific embodiment, the eye cell includes a corneal endothelialcell of a primate.

In a specific embodiment, the eye cell includes a human cornealendothelial cell.

In a specific embodiment, the eye cell is in the confluent state.

In one aspect, the present invention provides an agent for suppressingdifferentiation and/or promoting proliferation of a cell, comprising anagent suppressing GPR49/LGR5.

In one embodiment, the present invention provides an agent forsuppressing differentiation and/or promoting proliferation of a cellselected from an eye cell, a nerve cell including a cell derived from anneural crest cell (including corneal endothelial cell), and anepithelial cell such as a conjunctival epithelial cell, an amnioticepithelial cell, an oral mucosal epithelial cell, a nasal mucosalepithelial cell, and a corneal epithelial cell, comprising an agentsuppressing GPR49/LGR5.

In another embodiment, the present invention provides an agent forsuppressing differentiation and/or promoting proliferation of an eyecell, comprising an agent suppressing GPR49/LGR5.

In another embodiment, the agent suppressing GPR49/LGR5 is a nucleicacid, an antibody or an antibody fragment, or a functional equivalentthereof.

In another embodiment, the eye cell is a cell which does not proliferatein the stationary state.

In a specific embodiment, the eye cell includes at least one kind cellselected from a retinal cell, a vitreous body cell, a corneal epithelialcell, a corneal parenchymal cell and a corneal endothelial cell.

In a specific embodiment, the eye cell includes a corneal endothelialcell.

In a specific embodiment, the eye cell includes a corneal endothelialcell of a primate.

In a specific embodiment, the eye cell includes a human cornealendothelial cell.

In a specific embodiment, the eye cell is in the confluent state.

In a specific embodiment, the eye cell is provided in the form of acorneal tissue.

In a further aspect, the present invention provides a composition forpreserving the cornea or culturing a corneal endothelial cell,comprising the agent for suppressing differentiation and/or promotingproliferation of the present invention.

In a further aspect, the present invention provides a pharmaceuticalcomposition for treating a corneal endothelial cell disorder orpreventing the progression of a corneal endothelial cell disorder,comprising the agent for suppressing differentiation and/or promotingproliferation of the present invention.

In a further aspect, the present invention provides a therapeutic agentor a progression preventive agent for a corneal endothelial celldisorder, comprising a corneal endothelial cell which is cultured usingthe agent for suppressing differentiation and/or promoting proliferationdescribed in the present invention.

In one embodiment, the cell exists as a population having cell densityhigher than that of a normal corneal endothelial cell and/or containingundifferentiated cells in a larger amount.

In a further another aspect, the present invention provides a marker foridentifying a cell having a high proliferation ability among cornealendothelial cells and/or the differentiation ability of a cornealendothelial cell, comprising GPR49/LGR5.

In one embodiment of the marker of the present invention, the cellhaving a high proliferation ability is an undifferentiated cell.

In another embodiment of the marker of the present invention, the cellhaving a high proliferation ability is a stem cell.

In another embodiment of the marker of the present invention, thecorneal endothelial cell is a human cell.

In another embodiment of the marker of the present invention, theproliferation ability of the corneal endothelial cell is identified by acharacteristic selected from the group consisting of colony formingability, Ki-67 positivity and BrdU positivity.

In another aspect, the present invention provides a culture which is acorneal endothelial culture, in which the corneal endothelium exits atdensity higher than the cell density in the confluent state. The cornealendothelial culture in this case usually refers to a culture existing ina state which is different from the state existing in a living body.

In one embodiment, the cell density is about 570 cells/mm² or more.

In another embodiment, the cell density is about 700 cells/mm² or more.

In another embodiment, the cell density is about 800 cells/mm² or more.

In another embodiment, the cell density is about 1000 cells/mm² or more.

In another aspect, the present invention provides a corneal tissuecomprising a corneal endothelial cell, wherein a Ki67-positive cell inthe tissue exists at a ratio higher than the ratio in a living body,and/or the density of the corneal endothelial cell is higher than thedensity in a living body.

In one embodiment, the Ki67-positive cell exists at a ratio of about 4%or more.

In another embodiment, the Ki67-positive cell exists at a ratio of about7% or more.

In another embodiment, the Ki67-positive cell exists at a ratio of about10% or more.

In another embodiment, the density of the corneal endothelial cell isabout 4000 cells/mm² or more.

In another embodiment, the density of the corneal endothelial cell isabout 4500 cells/mm² or more.

In another embodiment, the density of the corneal endothelial cell isabout 5000 cells/mm² or more.

In another aspect, the present invention provides a method for usingGPR49/LGR5 as an index for identifying a cell having a highproliferation ability among corneal endothelial cells and/or thedifferentiation ability of a corneal endothelial cell.

In one embodiment in the method for using GPR49/LGR5 as an index of thepresent invention, the cell having a high proliferation ability is anundifferentiated cell.

In another embodiment in the method for using GPR49/LGR5 as an index ofthe present invention, the cell having a high proliferation ability is astem cell.

In another embodiment in the method for using GPR49/LGR5 as an index ofthe present invention, the corneal endothelial cell is a human cell.

In another embodiment in the method for using GPR49/LGR5 as an index ofthe present invention, the proliferation ability of the cornealendothelial cell is identified by a characteristic selected from thegroup consisting of colony forming ability, Ki-67 positivity and BrdUpositivity.

In a further another aspect, the present invention provides a detectionagent for identifying a cell having a high proliferation ability amongcorneal endothelial cells and/or the differentiation ability of acorneal endothelial cell, comprising a substance binding to GPR49/LGR5.

In one embodiment of the detection agent of the present invention, thedirection agent is an antibody or a fragment or functional equivalentthereof, or a nucleic acid primer or a probe.

In another embodiment of the detection agent of the present invention,the detection agent is labeled.

In another embodiment of the detection agent of the present invention,the cell having a high proliferation ability is a stem cell.

In another embodiment of the detection agent of the present invention,the corneal endothelial cell is a human cell.

In another embodiment of the detection agent of the present invention,the proliferation ability of the corneal endothelial cell is identifiedby a characteristic selected from the group consisting of colony formingability, Ki-67 positivity and BrdU positivity.

In a further another aspect, the present invention provides a marker foridentifying a cell having a high proliferation ability among cornealendothelial cells and/or the differentiation ability of a cornealendothelial cell, comprising SHH.

In one embodiment of the marker of the present invention, the cellhaving a high proliferation ability is an undifferentiated cell.

In another embodiment of the marker of the present invention, the cellhaving a high proliferation ability is a stem cell.

In another embodiment of the marker of the present invention, thecorneal endothelial cell is a human cell.

In another embodiment of the marker of the present invention, theproliferation ability of the corneal endothelial cell is identified by acharacteristic selected from the group consisting of colony formingability, Ki-67 positivity and BrdU positivity.

In another aspect, the present invention provides a method for using SHHas an index for identifying a cell having a high proliferation abilityamong corneal endothelial cells and/or the differentiation ability of acorneal endothelial cell.

In one embodiment in the method for using SHH as an index of the presentinvention, the cell having a high proliferation ability is anundifferentiated cell.

In another embodiment in the method for using SHH as an index of thepresent invention, the cell having a high proliferation ability is astem cell.

In another embodiment in the method for using SHH as an index of thepresent invention, the corneal endothelial cell is a human cell.

In another embodiment in the method for using SHH as an index of thepresent invention, the proliferation ability of the corneal endothelialcell is identified by a characteristic selected from the groupconsisting of colony forming ability, Ki-67 positivity and BrdUpositivity.

In a further another aspect, the present invention provides a detectionagent for identifying a cell having a high proliferation ability amongcorneal endothelial cells and/or the differentiation ability of acorneal endothelial cell, comprising a substance binding to SHH.

In one embodiment of the detection agent of the present invention, thedetection agent is an antibody or a fragment or functional equivalentthereof, or a nucleic acid primer or a probe.

In another embodiment of the detection agent of the present invention,the detection agent is labeled.

In another embodiment of the detection agent of the present invention,the cell having a high proliferation ability is a stem cell.

In another embodiment of the detection agent of the present invention,the corneal endothelial cell is a human cell.

In another embodiment of the detection agent of the present invention,the proliferation ability of the corneal endothelial cell is identifiedby a characteristic selected from the group consisting of colony formingability, Ki-67 positivity and BrdU positivity.

In a further another aspect, the present invention provides a marker foridentifying a cell having a high proliferation ability among cornealendothelial cells and/or the differentiation ability of a cornealendothelial cell, comprising a factor of the Hedgehog pathway.

In one embodiment of the marker of the present invention, the cellhaving a high proliferation ability is an undifferentiated cell.

In another embodiment of the marker of the present invention, the cellhaving a high proliferation ability is a stem cell.

In another embodiment of the marker of the present invention, thecorneal endothelial cell is a human cell.

In another embodiment of the marker of the present invention, theproliferation ability of the corneal endothelial cell is identified byat least one characteristic selected from the group consisting of colonyforming ability, Ki-67 positivity and BrdU positivity.

In another embodiment of the marker of the present invention, the factorof the Hedgehog pathway is selected from the group consisting of SHH,PTCT1, GLI1 and GLI2.

In a further another aspect, the present invention provides a method forusing a factor of the Hedgehog pathway as an index for identifying acell having a high proliferation ability among corneal endothelial cellsand/or the differentiation ability of a corneal endothelial cell.

In one embodiment of the method of the present invention, the cellhaving a high proliferation ability is an undifferentiated cell.

In another embodiment of the method of the present invention, the cellhaving a high proliferation ability is a stem cell.

In another embodiment of the method of the present invention, thecorneal endothelial cell is a human cell.

In another embodiment of the present invention, the proliferationability of the corneal endothelial cell is identified by at least onecharacteristic selected from the group consisting of colony formingability, Ki-67 positivity and BrdU positivity.

In another embodiment of the present invention, the factor of theHedgehog pathway is selected from the group consisting of SHH, PTCH1,GLI1 and GLI2.

In another aspect, the present invention provides a detection agent foridentifying a cell having a high proliferation ability among cornealendothelial cells and/or the differentiation ability of a cornealendothelial cell, comprising a substance binding to a factor of theHedgehog pathway.

In one embodiment of the detection agent of the present invention, thedetection agent is an antibody or a fragment or functional equivalentthereof, or a nucleic acid primer or a probe.

In another embodiment of the detection agent of the present invention,the detection agent is labeled.

In another embodiment of the detection agent of the present invention,the cell having a high proliferation ability is a stem cell.

In another embodiment of the detection agent of the present invention,the corneal endothelial cell is a human cell.

In another embodiment of the detection agent of the present invention,the proliferation ability of the corneal endothelial cell is identifiedby at least one characteristic selected from the group consisting ofcolony forming ability, Ki-67 positivity and BrdU positivity.

In another embodiment of the detection agent of the present invention,the factor of the Hedgehog pathway is selected from the group consistingof SHH, PTCH1, GLI1 and GLI2.

In another aspect, the present invention provides a marker foridentifying a cell having a high proliferation ability among cornealendothelial cells and/or the differentiation ability of a cornealendothelial cell, comprising a factor of the Wnt pathway.

In one embodiment of the marker of the present invention, the cellhaving a high proliferation ability is an undifferentiated cell.

In another embodiment of the marker of the present invention, the cellhaving a high proliferation ability is a stem cell.

In another embodiment of the marker of the present invention, thecorneal endothelial cell is a human cell.

In another embodiment of the marker of the present invention, theproliferation ability of the corneal endothelial cell is identified byat least one characteristic selected from the group consisting of colonyforming ability, Ki-67 positivity and BrdU positivity.

In another embodiment of the marker of the present invention, the factorof the Wnt pathway is selected from the group consisting of LRP6 andβ-catenin.

In another aspect, the present invention provides a method for using afactor of the Wnt pathway as an index for identifying a cell having ahigh proliferation ability among corneal endothelial cells and/or thedifferentiation ability of a corneal endothelial cell.

In one embodiment of the method of the present invention, the cellhaving a high proliferation ability is an undifferentiated cell.

In another embodiment of the method of the present invention, the cellhaving a high proliferation ability is a stem cell.

In another embodiment of the method of the present invention, thecorneal endothelial cell is a human cell.

In another embodiment of the method of the present invention, theproliferation ability of the corneal endothelial cell is identified by acharacteristic selected from the group consisting of colony formingability, Ki-67 positivity and BrdU positivity.

In another embodiment of the method of the present invention, the factorof the Wnt pathway is selected from the group consisting of LRP6 andβ-catenin.

In another aspect, the present invention provides a detection agent foridentifying a cell having a high proliferation ability among cornealendothelial cells and/or the differentiation ability of a cornealendothelial cell, comprising a substance binding to a factor of the Wntpathway.

In one embodiment of the detection agent of the present invention, thedetection agent is an antibody or a fragment or functional equivalentthereof, or a nucleic acid primer or a probe.

In another embodiment of the detection agent of the present invention,the detection agent is labeled.

In another embodiment of the detection agent of the present invention,the cell having a high proliferation ability is a stem cell.

In another embodiment of the detection agent of the present invention,the corneal endothelial cell is a human cell.

In another embodiment of the detection agent of the present invention,the proliferation ability of the corneal endothelial cell is identifiedby a characteristic selected from the group consisting of colony formingability, Ki-67 positivity and BrdU positivity.

In another embodiment of the detection agent of the present invention,the factor of the Wnt pathway is selected from the group consisting ofLRP6 and β-catenin.

In a further another aspect, the present invention provides adiagnostic, a detection kit, a diagnostic kit, a detection system, adiagnostic system or the like using the detection agent, marker or thelike of the present invention.

In a further another aspect, the present invention provides a treatmentmethod, a prevention method, use or the like using the pharmaceuticalcomposition, therapeutic agent or progression preventive agent of thepresent invention.

It is understood that the aforementioned characteristics can be used infurther combining one or more of the aforementioned characteristicstherewith.

A person skilled in the art recognizes still further embodiments andadvantages of the present invention by reading and understanding thefollowing detailed description, if necessary.

Advantages of the Invention

The present invention can identify the differentiation ability of a cellexisting in corneal endothelial cells and can identify a cell having ahigh proliferation ability, and as a result, it has become possible toeffectively identify and collect an undifferentiated cell which existsin the corneal endothelium at a small amount. In addition, the cornealendothelium can proliferate by using R-spondins, and a disease ordisorder of the corneal endothelium, which has previously beenimpossible or difficult to treat or prevent, can be treated orprevented.

BRIEF DESCRIPTION OF THE DRAWINGS

[FIG. 1] FIG. 1 shows the structure of GRP49/LGR5 (see Barker N. et al.,Gastroenterology, 2010 May; 138 (5) : 1681-96).

[FIG. 2-1] FIG. 2 shows expression of GPR49/LGR5 in the human cornea. a.shows, from the left side, the immunostaining in a human corneal sliceof GPR49/LGR5, nestin and ABCG2. The scale bar is 100 μm. In eachphotograph, from the upper side, parts corresponding to epithelium(Epi), stroma (Str) and endothelium (End) are shown. b. shows, from theleft side, the real time PCR of GPR49/LGR5, nestin and ABCG2. Thevertical axis shows the relative level of mRNA, when the level ofcorneal endothelium is set to be 1. N=4. Epi: corneal epithelium; Str:corneal stroma; End: corneal endothelium. The error bar shows S.E. *InStudent's t-test, p<0.05.

[FIG. 2-2] FIG. 2 shows expression of GPR49/LGR5 in the human cornea. c.shows a whole mount of immunostaining of GPR49/LGR5. Center shows acentral portion, and Periphery shows a peripheral portion. d. shows anenlarged view of the central region vs. peripheral portion region of thehuman cornea. Center shows a central portion, and Periphery shows aperipheral portion. e. shows the real time PCR of GPR49/LGR5 at acentral portion (Center) and a peripheral portion (Periphery) of thehuman cornea. The vertical axis shows the relative level of mRNA, whenthe level of corneal endothelium is set to be 1. N=3. The error barshows S.E. *In Student's t-test, p<0.05.

[FIG. 3-1] FIG. 3 shows expression of GPR49/LGR5 in a cultured cornealendothelial cell, a. shows the immunostaining of GPR49/LGR5 and nestinin a cultured human corneal endothelial cell (cHCEC). From the leftside, a phase contrast image, a GPR49 PI image and a nestin PI image areshown. From the upper side, in vivo, primary culturing (P0), and passagefirst generation (P1) are shown. The scale bar shows 100 μm.

[FIG. 3-2] FIG. 3 shows expression of GPR49/LGR5 in a cultured cornealendothelial cell. b. shows, from the left side, expressions in cHCEC ofGPR49/LGR5 and a nestin mRNA. The vertical axis shows the relative levelof mRNA, when the level of primary culturing (P0) is set to be 1. Invivo, and primary culturing (P0) are shown. N=4. An error bar shows S.E.*In Student's t-test, p<0.05. ND=not detected.

[FIG. 3-3] FIG. 3 shows expression of GPR49/LGR5 in a cultured cornealendothelium cell. c. shows the immunostain of GPR49/LGR5 in a culturedmonkey corneal endothelial cell (cMCEC). From the left side, a phasecontrast image and a GPR49 PI image are shown. From the upper side, invivo, primary culturing (P0), and passage first generation (P1) areshown. The scale bar shows 100 μm.

[FIG. 3-4] FIG. 3 shows expression of GPR49/LGR5 in a cultured cornealendothelium cell. d. shows expression in cMCEC of GPR49/LGR5 mRNA. Thevertical axis shows the relative level of mRNA, when the level ofprimary culturing (P0) is set to be 1. In vivo, primary culturing (P0),passage first generation (P1) and passage second generation (P2) areshown. N=3. An error bar shows S.E. *In Student's t-test, p<0.05.

[FIG. 4-1] FIG. 4 shows the characterization of a GPR49/LGR5-positivecell, a. shows the phase contrast of a GPR49/LGR5-positive cell (GPR49⁺)after cell sorting on the left side, and shows the phase contrast of anegative cell (GPR49⁻) on the right side. The scale bar shows 100 μm.

[FIG. 4-2] FIG. 4 shows the characterization of a GPR49/LGR5-positivecell. b. shows the surface area of each cell. The average cell size ofGPR49⁺ is 184.6±45.8 μm², and the average cell size of GPR49⁻ is326.78±78.8 μm². N=35. An error bar shows S.E. *In Student's t-test,p<0.01. c. shows the Ki-67 positivity ratio of GPR49⁺ and GPR49⁻. N=4.An error bar shows S.E. *In Student's t-test, p<0.05.

[FIG. 4-3] FIG. 4 shows the characterization of a GPR49/LGR5-positivecell. d. shows the cell sorting of the cell proliferation of GPR49⁺ bydouble staining (vertical axis: Cy3-Ki67 and transverse axis:FITC-GPR49): GPR49⁺/Ki-67⁺; 3.4%, GPR49⁺/Ki-67⁻; 3.8%, GPR49⁻/Ki-67⁻;0%, GPR49⁻/Ki-67⁻; 92.8%.

[FIG. 5-1] FIG. 5 shows the Hedgehog transmission pathway in cHCEC. a.shows expression of a Hedgehog signal-associated gene (In the upper rowand from the left side, Shh, Smo and Ptch1, and in the lower row andfrom the left side, Gli1 and Gli2). The vertical axis shows the relativelevel of mRNA, when the level of center is set to be 1.

[FIG. 5-2] FIG. 5 shows the Hedgehog signal transmission pathway incHCEC. b. shows, from the left side, the immunostaining of a control,GPR49/LGR5 and Ki-67 in HCEC treated with 100 ng/ml rhShh (second fromthe left side), 10 μM purmorphamine (Pur) (third from the left side) and10 μM cyclopamine (Cyc) (the rightmost). The upper row shows GPR49staining, and the lower row shows Ki67 staining. The control is 0.1%DMSO. The scale bar shows 100 μm.

[FIG. 5-3] FIG. 5 shows the Hedgehog signal transmission pathway incHCEC. c. shows, in the upper row and from the left side, the real timePCR of GPR49/LGR5 and Ptch1, and in the lower row and from the leftside, the real time PCR of Gli1 and Gli2. Control means a control, andthe vertical axis shows the relative level of mRNA, when the result fromthe control is set to be 1. rhShh shows treatment with rhShh, Pur showstreatment with purmorphamine, and Cyc shows treatment with cyclopamide.N=4. An error bar shows S.E. *In Student's t-test, p<0.05. **InStudent's T-test, p<0.01.

[FIG. 5-4] FIG. 5 shows the Hedgehog signal transmission pathway incHCEC. d. shows expression (relative mRNA level) of GPR49/LGR5 in cHCECtreated with rhSHH. e. shows the immunostaining of GPR49/LGR5 in cHCECtreated with rhSHH. The scale bar shows 100 μm.

[FIG. 5-5] FIG. 5 shows the Hedgehog signal transmission pathway incHCEC. f. shows, from the left side, the immunostaining of a control,Ki-67 in cHCEC treated with 100 ng/ml rhSHH, 2 μM Pur and 2 μM Cyc. Theupper row shows GRP49 staining. The lower row shows Ki67 staining. Thecontrol is 0.01% DMSO. The scale bar shows 100 μm.

[FIG. 5-6] FIG. 5 shows the Hedgehog signal transmission pathway incHCEC. g. shows, from the light side, the Ki-67 positivity ratio of acontrol, cHCEC treated with, 100 ng/ml rhShh, 2 μM purmorphamine (Pur)and 2 μM cyclophosphamide (Cyc). N=5. An error bar shows S.E. *InStudent's t-test, p<0.01.

[FIG. 6-1] FIG. 6 shows the function of GPR49/LGR5 in a cornealendothelial cell. a. shows the knock down effect of GPR49/LGR5 shRNA.The vertical axis shows the relative level of mRNA, when the level of acontrol (NT) is set to be 1. From the left side, the effect on GPR49with NT (control), shGPR49-587, shGPR49-588, and shGPR49-589 is shown.

[FIG. 6-2] FIG. 6 shows the function of GPR49/LGR5 in a cornealendothelial cell. b. shows expressions of Ptch1, Gli1 and Gli2 (from theleft side) treated with shRNA (589). NT: non-target. N=5. An error barshows S.E. ND=not detected.

[FIG. 6-3] FIG. 6 shows the function of GPR49/LGR5 in a cornealendothelial cell. c. shows the effect of overexpression of GPR49/LGR5 oncHCEC. The left side shows a phase contrast image, and the right sideshows Na⁺/K⁺ ATPase PI staining. The upper row shows GPR49 expression,and the lower row shows NT (control).

[FIG. 6-4] FIG. 6 shows the function of GPR49/LGR5 in a cornealendothelial cell. d. shows, in the upper row and from the left side,expressions of GPR49/LGR5 and Ptch1, and in the lower row and from theleft side, expressions of Gli1 and Gli2 mRNA. NT shows a control, andExpGPR49 shows a GPR49/LGR5 expression product. N=3. An error bar showsS.E. *In Student's t-test, *p<0.01.

[FIG. 6A] FIG. 6A shows the action of shLGR5 in a human cornealendothelial cell (CDC). (A) shows, from the left side, real time PCRconcerning GPR49/LGR5, Ptch1, Gli1 and Gli2 in NT and shLGR-transfectedcell. Average±SEM. **P<0.05. N=3. (A) represents the graphs shown inFIG. 6-2 together with GPR49/LGR5, and is partially overlapped. (B)shows a phase contrast microscope image (left column) of Ki67 and theimmunostain (light column) of Ki67 in NT (upper row) andshLGR-transfected human CEC (lower row). An arrow shows a Ki67(+) cell.The scale bar is 100 μm. In (B), the right graph shows the results ofimplementation of immunocytochemistry study concerning percentage of aKi67 cell in order to demonstrate influence of GPR49/LGR5 gene knockdown on CEC proliferation. The left side shows a control (NT), and theright side shows a shLGR5-treated cell (shLGR5).

[FIG. 6B] FIG. 6B shows the functions of GPR49/LGR5 and RSPO1 in acorneal endothelial cell (CEC). (A) shows a phase contrast microscopeimage (the leftmost), as well as the immunostaining of GPR49/LGR5(secondfrom the left side), Na⁺/K⁺ ATPase (second from the right side) and ZO1(the rightmost) in NT (the upper row) and shLGR-transfected human CEC(the lower row). The scale bar=100 μm. FIG. 6B (A) is partiallyoverlapped with FIG. 6-3 (phase contrast image, NaKATP), but forcomparison, both of them are shown side by side. (B) shows the celldensity of CEC in the presence or absence of RSPO1. Average±SEM.**P<0.01. N=5. (C) shows the cell density of NT and LGR5-transfectedcell. Average±SEM. **P<0.01. N=5. (D) shows real time PCR concerningEMT-associated genes (Snail (the leftmost), Slug (second from the leftside), Twist (second from the right side) and collagen 1 (therightmost)) in NT and a LGR5-transfected cell. Average±SEM. **P<0.01.N=3. (E) shows a phase contrast microscope image of human CEC in thepresence (RSPO1(+), right) or absence (RSPO1(−), left) of RSPO1 (50ng/ml). The scale bar=100 μm. (F) shows the western blotting ofactivated β-catenin (the uppermost row), pLRP6 (second from the upperside), tLRP6 (second from the lower side) and β-actin (the lowermostrow) in NT and a LGR5-transfected cell in the presence or absence ofRSPO1 (50 ng/ml).

[FIG. 6C] FIG. 6C shows expression and function of RSPO in a humancorneal endothelial cell (CEC). (A) the upper panel shows the results ofPCR concerning RSPO1 (the uppermost row), RSPO2 (second row from theupper side), RSPO3 (third row from the upper side) and RSPO4 (fourth rowfrom the upper side) in a human corneal epithelial cell (Epi), aparenchymal cell (Stroma) and an endothelial cell (Endo). The lowerpanel shows the results of PCR concerning RSPO1 (the uppermost row),RSPO2 (second row from the upper side), RSPO3 (third row from the upperside) and RSPO4 (fourth row from the upper side) at a central part(Center) and a periphery part (Periphery), among endothelial cells(Endo) (B) shows a phase contrast microscope image of cultured human CECin the presence or absence of RSPO1 (the uppermost row), RSPO2 (secondrow from the upper side), RSPO3 (third row from the upper side) andRSPO4 (fourth row from the upper side) (all are 50 ng/ml). The scalebar=100 μm. (C) shows the immunostaining of Ki67 in human CEC in thepresence or absence of RSPO1 (the uppermost row), RSPO2 (second row fromthe upper side), RSPO3 (third row from the upper side) and RSPO4 (fourthrow from the upper side) (all are 50 ng/ml). The scale bar shows 100 μm.

[FIG. 6D] FIG. 6D shows a schematic view of the molecular mechanism ofCEC maintenance. Human CEC shows regional diversity regarding GRP49/LGR5expression. GRP49/LGR5 is expressed peculiarly at a peripheral region ofCEC, and herein, HH signal transmission is clearly activated. GRP49/LGR5is a target molecule in the HH pathway, and under in vitro conditions,the HH pathway could induce CEC proliferation. However, under in vivocircumstances, stimulation of only the HH pathway was insufficient forinducing CEC proliferation. The permanent expression of GRP49/LGR5maintained a normal CEC phenotype by inhibition of the Wnt pathway.RSPO1 which is a GRP49/LGR5 ligand accelerated CEC proliferation invivo, and inhibited MT via the Wnt pathway.

[FIG. 7] FIG. 7 shows an outline of R-spondins 1 to 4.

[FIG. 8] FIG. 8 shows the detection (magnification×200) of aproliferating cell using a Click-iT EdU Imaging kit in Example 9. Monkeycultured corneal endothelial cells were seeded, the cells were exchangedwith a medium containing R-spondin 1 (from the left side, 0, 10, 50ng/ml) in the state where the cells were in the approximately confluent,and after 24 hours from the addition of R-spondin 1, EdU was stainedwith Click-iT EdU, and an EdU-positive cell rate was counted.

[FIG. 9] FIG. 9 shows an EdU-positive cell ratio in FIG. 8. As a resultof counting of EdU-positive cell/DAPI, it was found that theEdU-positive cell was increased two times in a well to which R-spondin 1was added at 10 ng/ml or 50 ng/ml, as compared with a control.

[FIG. 10] FIG. 10 shows the endothelial cell density of cornealendothelial cells which are cultured by the method shown in FIG. 8. Aphotograph was taken with a phase contrast microscope, and the celldensity was measured using a corneal endothelial cell densitycalculating software Konan Storage system KSS-400EB. From the left side,addition of no medium, 10 ng/ml R-spondin 1, and 50 ng/ml R-spondin 1are shown.

[FIG. 11] FIG. 11 shows the action of R-spondin 1 in a human culturedcorneal endothelial cell. A Ki67-positive cell rate was increased in acorneal endothelial cell to which RSPO1 was added (50 ng/ml (the rightupper side), 500 ng/ml (the left lower side)), as compared with acontrol. The left upper side shows a control, the right upper side showsan example of culturing with 50 ng/ml R-spondin 1, the left lower sideshows an example of culturing with 500 ng/ml R-spondin 1, and the rightlower side shows an example of culturing with 1000 ng/ml R-spondin 1.Ki67 is stained with green, a color is not seen in the control, Ki67 isremarkably stained at 50 ng/ml and 500 ng/ml, and staining is alsoobserved at 1000 ng/ml. PI is stained with red, and all cells arestained.

[FIG. 12] FIG. 12 shows the action of R-spondin 2 in a human culturedcorneal endothelial cell. In a corneal endothelial cell to which RSPO2was added, a Ki67-positive cell rate was slightly increased as comparedwith a control. The left side shows an example of culturing with 50ng/ml R-spondin 2, the right side shows an example of culturing with 100ng/ml R-spondin 2, the upper side shows limbus cornea and the lower sideshows a central part.

[FIG. 13] FIG. 13 shows the action of R-spondin 3 in a human culturedcorneal endothelial cell. In a corneal endothelial cell to which RSPO3was added, a Ki67-positive cell rate was slightly increased as comparedwith a control. The left side shows an example of culturing with 50ng/ml R-spondin 3, the right side shows an example of culturing with 200ng/ml R-spondin 3, the upper side shows limbus cornea, and the lowerside shows a central part.

[FIG. 14] FIG. 14 shows the action of R-spondin 4 in a human culturedcorneal endothelial cell. In a corneal endothelial cell to which RSPO4was added, a Ki67-positive cell rate was slightly increased as comparedwith a control. The left side shows an example of culturing with 50ng/ml R-spondin 4, the right side shows an example of culturing with 500ng/ml R-spondin 4, the upper side shows limbus cornea, and the lowerside shows a central part.

[FIG. 15] FIG. 15 shows the results obtained by adding R-spondin 1 to amedium at a concentration of 10 ng/ml at the time point when thesubculture of human corneal endothelial cells was performed, thereafter,the cells reached confluent, and were cultured for 2 weeks or longer,and clear change was not recognized in the corneal endothelial density;continuing culturing; observing a cell form with a phase contrastmicroscope; and calculating the cell density. A photograph shows thenumber of days after addition (from the left side, 0 day, 7 day, 14 dayand 21 day). The lower graph shows a change in the cell density of thegroup of R-spondin 1 addition on 0 day, 7 day, 14 day and 21 day, ascompared with a non-addition group.

[FIG. 16] FIG. 16 shows the results concerning organ culturing using arabbit sclerocornea slice, which are obtained by culturing the organ inan incubator at 37° C. for one week in DMEM (INVITROGEN, catalog number:12320)+10% fetal bovine serum (FBS) (BIOWEST, catalog number: S1820-500)in the presence (from the right side, 100 ng/ml, 10 ng/ml, 1 ng/ml) orabsence (the leftmost, control) of R-spondin 1; immunostaining a cornealendothelial cell using Ki67 (Sigma-Aldrich Co., catalog number: P6834)as a marker of cell proliferation; and observing the resultant with afluorescent microscope. Nuclear staining was performed with DAPI, aKi67-positive cell rate was calculated, and the results are shown in theleft lower panel. In addition, immunostaining was performed with ZO-1,and the cell density was calculated, and results are shown in the rightlower panel.

MODE FOR CARRYING OUT THE INVENTION

The present invention will be described below. It should be understoodthat expression in a singular form also includes the conception of itsplural form, unless specifically defined, throughout the presentdescription. Therefore, it should be understood that an article in asingular form (e.g., “a”, “an”, and “the” in English) also includes theconception of its plural form, unless specifically mentioned. Inaddition, it should be understood that terms used herein are used in asense which is usually used in the art, unless specifically mentioned.Therefore, unless defined otherwise, all technical terminology andscientific and technical terminology used herein have the same meaningsas those generally understood by a person skilled in the art to whichthe present invention belongs. In the case of confliction, the presentdescription (including definition) prevails.

As used herein, “GPR49/LGR5” is a seven-transmembrane protein which is amember of LGR family. GPR49/LGR5 is named based on a leucine richrepeat-containing G protein-coupled receptor-5, which is an abnormal Gprotein-coupled receptor (GPCR), that is characterized by a largeN-terminal extracellular domain containing leucine rich repeats that insome cases have been shown to be important for interaction withglycoprotein ligands, and recently, the frequency of such a name isincreased, and both names are described together herein. This is alsoknown as FEX HG38, GPR67. An amino acid sequence of human GPR49/LGR5 anda gene sequence encoding this are disclosed in NCBI registration numberNP_003658.1 (SEQ ID No.: 2) and NM_003667.2 (SEQ ID No.: 1),respectively. In the art, they are also called by a single name such asGPR49 or LGR5. As used herein, “GPR49/LGR5” is described, but it isunderstood that any expression shows the same meaning. GPR49/LGR5 can beidentified by the accession number of OMIM: 606667. It is understoodwhen used for the purpose of the present description, “GPR49/LGR5” meansnot only a protein having an amino acid sequence described in a specificsequence number or an accession number (or nucleic acid encoding thesame), but also a functionally active derivative thereof, a functionallyactive fragment thereof, or a homolog thereof, or a mutant encoded by anucleic acid which hybridizes with a nucleic acid encoding this proteinunder a high stringency condition or a low stringency condition.

In addition, the same shall apply to all other proteins listed in thepresent invention. Therefore, the already defined name of a protein or anucleic acid refers to not only a protein or a nucleic acid as shown inSequence Listing, but also a functionally active derivative, or afunctionally active fragment thereof, or a homolog thereof, or a mutantencoded by a nucleic acid which hybridizes with a nucleic acid encodingthe protein under a high stringency condition or a low stringencycondition, preferably under the aforementioned condition. Preferably, a“derivative” or an “analog of a constituent protein” or a “mutant” usedherein, does not intend limitation, but includes a molecule containing aregion substantially homologous to a constituent protein. In variousembodiments, such a molecule is at least 30%, 40%, 50%, 60%, 70%, 80%,90%, 95% or 99% identical with the comparative lengths of amino acidsequences, or in comparison with sequences which are aligned by acomputer homology program known in the art, or a nucleic acid encodingsuch a molecule can hybridize with a sequence encoding a constituentprotein under a stringent condition, under a moderately stringentcondition, or under not stringent condition. This is an outcome ofmodification of a naturally-occurring protein by amino acidsubstitution, deletion and addition, respectively, and means a proteinsuch that a derivative thereof still exhibits the biological function ofa naturally-occurring protein to a degree that may not be necessarilythe same. For example, it is also possible to investigate the biologicalfunction of such a protein by an appropriately utilizable in vitro assaywhich is described herein or known in the art. In the present invention,human GPR49/LGR5 is mainly discussed, but since it is known that manymammals other than human, such as chimpanzee (Pan troglodytes)(ENSPTRG00000005223 (XR_021586.1)), rhesus monkey (Macaca mulatta)(ENSMMUG00000020942), mouse (Mus musculus) (ENSMUSG00000020140), rat(Rattus norvegicus) (ENSRNOG00000004221 (LOC687868)), guinea pig (Caviaporcellus) (ENSCPOG00000009492), dog (Canis familiaris)(ENSCAFG00000000451), cat (Felis catus) (ENSFCAG00000008064), andchicken (Gallus gallas) (ENSGALG00000010163), express a GPR49/LGR5protein, it is understood that these mammals are within the scope of thepresent invention.

It has been found that GPR49/LGR5 forms a part of different proteincomplexes, which is involved in abnormal processing of APP with gammasecretase, in an Alzheimer's disease. Since it has been found thatGPR49/LGR5 is a part of an Aph1a complex, a Fe65L2 complex, an APP-C99complex and a BACE1 complex, GPR49/LGR5 can be also detected bydetecting these complexes. These complexes are named after respectiveimportant protein compounds, which are used as TAP technical entrypoints.

The “functionally active” as used herein refers to the polypeptide ofthe present invention, that is, a polypeptide having the structuralfunction, controlling function, or biochemical function, such as abiological activity, of a protein, which is a fragment or a derivative,in accordance with an aspect associated with a fragment or a derivative,as used herein.

In the present invention, the fragment of GPR49/LGR5 is a polypeptidecomprising an arbitrary region of GPR49/LGR5, and may not have thebiological function of natural GPR49/LGR5. Examples of the fragmentinclude fragments comprising an extracellular region of GPR49/LGR5. Theextracellular region of GPR49/LGR5 corresponds to 1-556^(th),615-637^(th), 704-722^(nd), and 792-800^(th) in the amino acid sequenceof SEQ ID No.: 2. A transmembrane region corresponds to 557-579^(th),592-614^(th), 638-660^(th), 681-703^(rd), 723-745^(th), 769-791^(st) and801-823^(rd) in the amino acid sequence of SEQ ID No.: 2.

A representative nucleotide sequence of GPR49/LGR5 can be:

(a) a polynucleotide having a nucleotide sequence of SEQ ID No.: 1 or afragment sequence thereof;

(b) a polynucleotide encoding a polypeptide consisting of an amino acidsequence of SEQ ID No.: 2 or a fragment thereof;

(c) a polynucleotide encoding a variant polypeptide in which one or moreamino acids have one mutation selected from the group consisting ofsubstitution, addition and deletion in an amino acid sequence of SEQ IDNo.: 2, or a fragment thereof, the variant polypeptide having abiological activity;

(d) a polynucleotide which is a splicing mutant or an allele mutant of anucleotide sequence of SEQ ID No.: 1 or a fragment thereof;

(e) a polynucleotide encoding a species homolog of a polypeptideconsisting of an amino acid sequence of SEQ ID No.: 2 or a fragmentthereof;

(f) a polynucleotide encoding a polypeptide wherein the polynucleotidehybridizes with the polynucleotide of any one of (a) to (e) under astringent condition, and the polypeptide has a biological activity; or

(g) a polynucleotide encoding a polypeptide wherein the polynucleotideconsists of a nucleotide sequence in which the identity to thepolynucleotide of any one of (a) to (e) or a complementary sequencethereof is at least 70%, and the polypeptide has a biological activity.Herein, the biological activity representatively refers to an activityof GPR49/LGR5.

An amino acid sequence of GPR49/LGR5 can be:

(a) a polypeptide consisting of an amino acid sequence of SEQ ID No.: 2or a fragment thereof;

(b) a polypeptide in which one or more amino acids have one mutationselected from the group consisting of substitution, addition anddeletion in an amino acid sequence of SEQ ID No.: 2, and the polypeptidehas a biological activity;

(c) a polypeptide encoded by a splicing mutant or an allele mutant of anucleotide sequence of SEQ ID No.: 1;

(d) a polypeptide which is a species homolog of an amino acid sequenceof SEQ ID No.: 2; or

(e) a polypeptide having an amino acid sequence in which the identity tothe polypeptide of any one of (a) to (d) is at least 70%, and having abiological activity. Herein, the biological activity representativelyrefers to an activity of GPR49/LGR5.

In a context with the present invention, the “substance binding toGPR49/LGR5” or the “GPR49/LGR5 interaction molecule” is a molecule or asubstance which binds to GPR49/LGR5 at least temporarily, andpreferably, can indicate that it has bound thereto (e.g., in the statewhere it is labeled or can be labeled). The substance binding toGPR49/LGR5 may be an inhibitor of GPR49/LGR5, and examples thereof mayinclude an antibody, an antisense-oligonucleotide, siRNA, a lowmolecular weight (LMW) molecule, a binding peptide, an aptamer, aribozyme and a peptidomimetic, and for example, a binding protein or abinding peptide directed to GPR49/LGR5, particularly, directed to anactive site of GPR49/LGR5, as well as a nucleic acid directed to aGPR49/LGR5 gene are also included. A nucleic acid to GPR49/LGR5 refersto, for example, double-stranded or single-stranded DNA or RNA whichinhibits expression of a GPR49/LGR5 gene or the activity of GPR49/LCR5,or a modified product or derivative thereof, and includes, withoutlimitation, an antisense nucleic acid, an aptamer, siRNA (low-molecularinterference RNA) and a ribozyme. As used herein, concerning GPR49/LGR5,the “binding protein” or the “binding peptide” refers to a kind of aprotein or a peptide which binds to GPR49/LGR5, and includes, but is notlimited to, a polyclonal antibody or a monoclonal antibody, an antibodyfragment and a protein skeleton directed to GPR49/LGR5.

As used herein, “R-spondin(s)” refers to a gene group having the samestructure and function as those of R-spondin 1 and the like, and isexplained in Non-Patent Document 4 (J. Yoon, J. Lee, Cellular Signalling24 (2012) 369-377). For example, it is known, as a structure, thatR-spondin(s) has a domain of SP, CR, TSR and BR from the N-terminal. Inaddition, as a function, it is known that R-spondin(s) is a ligand forGPR49. Therefore, R-spondins can be identified using such a structureand function as an index. For example, in human, R-spondin 1 (RSPO1OMIM: 609595; nucleic acid sequence (gene sequence): SEQ ID No.: 3, 35,37 or 39; amino acid sequence: SEQ ID No.: 4, 36, 38 or 40), R-spondin 2(RSPO2 OMIM: 610575, SEQ ID Nos.: 5 and 6), R-spondin 3 (RSPO3 OMIM:610574, SEQ ID Nos.: 7 and 8), R-spondin 4 (RSPO4 OMIM: 610573: nucleicacid sequence (gene sequence): SEQ ID Nos.: 9 and 41; amino acidsequence: SEQ ID No.: 10 or 42) are known. From the information ofNon-Patent Document 4, respective R-spondins are summarized as in FIG.7.

A representative nucleotide sequence of R-spondin 1 can be:

(a) a polynucleotide having a nucleotide sequence of SEQ ID No.: 3, 35,37 or 39, or a fragment sequence thereof;

(b) a polynucleotide encoding a polypeptide consisting of an amino acidsequence of SEQ ID No.: 4, 36, 38 or 40, or a fragment thereof;

(c) a polynucleotide encoding a variant polypeptide in which one or moreamino acids have one mutation selected from the group consisting ofsubstitution, addition and deletion in an amino acid sequence of SEQ IDNo.: 4, 36, 38 or 40, or a fragment thereof, the variant polypeptidehaving a biological activity;

(d) a polypeptide which is a splicing mutant or an allele mutant of anucleotide sequence of SEQ ID NO.: 3, 35, 37 or 39, or a fragmentthereof;

(e) a polynucleotide encoding a species homolog of a polypeptideconsisting of an amino acid sequence of SEQ ID No.: 4, 36, 38 or 40, ora fragment thereof;

(f) a polynucleotide encoding a polypeptide wherein the polynucleotidehybridizes with the polynucleotide of any one of (a) to (e) under astringent condition, and the polypeptide has a biological activity; or

(g) a polynucleotide encoding a polypeptide wherein the polynucleotideconsists of a nucleotide sequence in which the identity to thepolynucleotide of any one of (a) to (e) or a complementary sequencethereof is at least 70%, and the polypeptide has a biological activity.Herein, the biological activity representatively refers to an activityof R-spondin 1.

An amino acid sequence of R-spondin 1 can be:

(a) a polypeptide consisting of an amino acid sequence of SEQ ID No.: 4,36, 38 or 40, or a fragment thereof;

(b) a polypeptide in which one or more amino acids have one mutationselected from the group consisting of substitution, addition anddeletion in an amino acid sequence of SEQ ID No.: 4, 36, 38 or 40, andthe polypeptide has a biological activity;

(c) a polypeptide encoded by a splicing mutant or an allele mutant of anucleotide sequence of SEQ ID No.: 3, 35, 37 or 39;

(d) a polypeptide which is a species homolog of an amino acid sequenceof SEQ ID No.: 4, 36, 38 or 40; or

(e) a polypeptide having an amino acid sequence in which the identity tothe polypeptide of any one of (a) to (d) is at least 70%, and having abiological activity. Herein, the biological activity representativelyrefers an activity of R-spondin 1.

A representative nucleotide sequence of R-spondin 2 can be:

(a) a polynucleotide having a nucleotide sequence of SEQ ID No.: 5 or afragment sequence thereof;

(b) a polynucleotide encoding a polypeptide consisting of an amino acidsequence of SEQ ID No.: 6 or a fragment thereof;

(c) a polynucleotide encoding a variant polypeptide in which one or moreamino acids have one mutation selected from the group consisting ofsubstitution, addition and deletion in an amino acid sequence of SEQ IDNo: 6, or a fragment thereof, the variant polypeptide having abiological activity;

(d) a polynucleotide which is a splicing mutant or an allele mutant of anucleotide sequence of SEQ ID No.: 5 or a fragment thereof;

(e) a polynucleotide encoding a species homolog of a polypeptideconsisting of an amino acid sequence of SEQ ID No.: 6 or a fragmentthereof;

(f) a polynucleotide encoding a polypeptide wherein the polynucleotidehybridizes with the polynucleotide of any one of (a) to (e) under astringent condition, and the polypeptide has a biological activity; or

(g) a polynucleotide encoding a polypeptide which consists of anucleotide sequence in which the identity to the polynucleotide of anyone of (a) to (e) or a complementary sequence thereof is at least 70%,and the polypeptide has a biological activity. Herein, the biologicalactivity representatively refers to an activity of R-spondin 2.

An amino acid sequence of R-spondin 2 can be:

(a) a polypeptide consisting of an amino acid sequence of SEQ ID No.: 6or a fragment thereof;

(b) a polypeptide in which one or more amino acids have one mutationselected from the group consisting of substitution, addition anddeletion in an amino acid sequence of SEQ ID No.: 6, and the polypeptidehas a biological activity;

(c) a polypeptide encoded by a splicing mutant or an allele mutant of anucleotide sequence of SEQ ID No.: 5;

(d) a polypeptide which is a species homolog of an amino acid sequenceof SEQ ID No.: 6; or

(e) a polypeptide having an amino acid sequence in which the identity tothe polypeptide of any one of (a) to (d) is at least 70%, and having abiological activity. Herein, the biological activity representativelyrefers to an activity of R-spondin 2.

A representative nucleotide sequence of R-spondin 3 can be:

(a) a polynucleotide having a nucleotide sequence of SEQ ID No.: 7 or afragment sequence thereof;

(b) a polynucleotide encoding a polypeptide consisting of an amino acidsequence of SEQ ID No.: 8 or a fragment thereof;

(c) a polynucleotide encoding a variant polypeptide in which one or moreamino acids have one mutation selected from the group consisting ofsubstitution, addition and deletion in an amino acid sequence of SEQ IDNo.: 8, or a fragment thereof, the variant polypeptide having abiological activity;

(d) a polynucleotide which is a splicing mutant or an allele mutant of anucleotide sequence of SEQ ID No.: 7 or a fragment thereof;

(e) a polynucleotide encoding a species homolog of a polypeptideconsisting of an amino acid sequence of SEQ ID No.: 8 or a fragmentthereof;

(f) a polynucleotide encoding a polypeptide which hybridizes with thepolynucleotide of any one of (a) to (e) under a stringent condition, andthe polynucleotide has a biological activity; or

(g) a polynucleotide encoding a polypeptide wherein the polynucleotideconsists of a nucleotide sequence in which the identity to thepolynucleotide of any one of (a) to (e) or a complementary sequencethereof is at least 70%, and the polypeptide has a biological activity.Herein, the biological activity representatively refers to an activityof R-spondin 3.

An amino acid sequence of R-spondin 3 can be:

(a) a polypeptide consisting of an amino acid sequence of SEQ ID No.: 8or a fragment thereof;

(b) a polypeptide in which one or more amino acids have one mutationselected from the group consisting of substitution, addition anddeletion in an amino acid sequence of SEQ ID No.: 8, and the polypeptidehas a biological activity;

(c) a polypeptide encoded by a splicing mutant or an allele mutant of anucleotide sequence of SEQ ID No.: 7;

(d) a polypeptide which is a species homolog of an amino acid sequenceof SEQ ID No.: 8; or

(e) a polypeptide having an amino acid sequence in which the identity tothe polypeptide of any one of (a) to (d) is at least 70%, and having abiological activity. Herein, the biological activity representativelyrefers to an activity of R-spondin 3.

A representative nucleotide sequence of R-spondin 4 can be:

(a) a polynucleotide having a nucleotide sequence of SEQ ID No.: 9 or41, or a fragment sequence thereof;

(b) a polynucleotide encoding a polypeptide consisting of an amino acidsequence of SEQ ID No.: 10 or 42, or a fragment thereof;

(c) a polynucleotide encoding a variant polypeptide in which one or moreamino acids have one mutation selected from the group consisting ofsubstitution, addition and deletion in an amino acid sequence of SEQ IDNo.: 10 or 42, or a fragment thereof, the variant polypeptide having aphysiological activity;

(d) a polynucleotide which is a splicing mutant or an allele mutant of anucleotide sequence of SEQ ID No.: 9 or 41, or a fragment thereof;

(e) a polynucleotide encoding a species homolog of a polypeptideconsisting of an amino acid sequence of SEQ ID No.: 10 or 42, or afragment thereof;

(f) a polynucleotide encoding a polypeptide wherein the polynucleotidehybridizes with the polynucleotide of any one of (a) to (e) under astringent condition, and the polypeptide has a biological activity; or

(g) a polynucleotide encoding a polypeptide wherein the polynucleotideconsists of a nucleotide sequence in which the identity to thepolynucleotide of any one of (a) to (e) or a complementary sequencethereof is at least 70%, and the polypeptide has a biological activity.Herein, the biological activity representatively refers to an activityof R-spondin 4.

An amino acid sequence of R-spondin 4 can be:

(a) a polypeptide consisting of an amino acid sequence of SEQ ID No.: 10or 42, or a fragment thereof;

(b) a polypeptide in which one or more amino acids have one mutationselected from the group consisting of substitution, addition anddeletion in an amino acid sequence of SEQ ID No.: 10 or: 42, and thepolypeptide has a biological activity;

(c) a polypeptide encoded by a splicing mutant or an allele mutant of anucleotide sequence of SEQ ID No.: 9 or 41;

(d) a polypeptide which is a species homolog of an amino acid sequenceof SEQ ID No.: 10 or 42; or

(e) a polypeptide having an amino acid sequence in which the identity tothe polypeptide of any one of (a) to (d) is at least 70%, and having abiological activity. Herein, the biological activity representativelyrefers to an activity of R-spondin 4.

As used herein, “SONIC HEDGEHOG (SHH)” is one of five kinds of proteinsbelonging to Hedgehog (HH) family, and a gene encoding this is shown byshh. SHH has a role in patterning many organs such as limbs and midlinestructures in the brain, as the most important morphogen in development.It is known that, in mutation of a human sonic hedgehog gene, deletionof ventral midline occurs to cause holoprosencephaly (HPE), andadditionally, hyperdactyly is generated due to a cause of change in acis regulating element. As other proteins of this family, there areDesert Hedgehog (DHH) and Indian Hedgehog (IHH) in a mammal. Forexample, SHH (SHH OMIM 600725; human: NM_000193 (SEQ ID Nos.: 11 and12); mouse: NM_009170) is known as a protein of human.

A representative nucleotide sequence of shh can be:

(a) a polynucleotide having a nucleotide sequence of SEQ ID No.: 11 or afragment thereof;

(b) a polynucleotide encoding a polypeptide consisting of an amino acidsequence of SEQ ID No.: 12 or a fragment thereof;

(c) a polynucleotide encoding a variant polypeptide in which one or moreamino acids have one mutation selected from the group consisting ofsubstitution, addition and deletion in an amino acid sequence of SEQ IDNo.: 12, or a fragment thereof, the variant polypeptide having abiological activity;

(d) a polynucleotide which is a splicing mutant or an allele mutant of anucleotide sequence of SEQ ID No.: 11 or a fragment thereof;

(c) a polynucleotide encoding a species homolog of a polypeptideconsisting of an amino acid sequence of SEQ ID No.: 12 or a fragmentthereof;

(f) a polynucleotide encoding a polypeptide wherein the polynucleotidehybridizes with the polynucleotide of any one of (a) to (e) under astringent condition, and the polypeptide has a biological activity; or

(g) a polynucleotide encoding a polypeptide wherein the polynucleotideconsists of a nucleotide sequence in which the identity to thepolynucleotide of any one of (a) to (e) or a complementary sequencethereof is at least 70%, and the polypeptide has a biological activity.Herein, the biological activity representatively refers to an activityof SHH.

An amino acid sequence of SHH can be:

(a) a polypeptide consisting of an amino acid sequence of SEQ ID No.: 12or a fragment thereof;

(b) a polypeptide in which one or more amino acids have one mutationselected from the group consisting of substitution, addition anddeletion in an amino acid sequence of SEQ ID No.: 12, and thepolypeptide has a biological activity;

(c) a polypeptide encoded by a splicing mutant or an allele mutant of anucleotide sequence of SEQ ID No.: 11;

(d) a polypeptide which is a species homolog of an amino acid sequenceof SEQ ID No.: 12; or

(e) a polypeptide having an amino acid sequence in which the identity tothe polypeptide of any one of (a) to (d) is at least 70%, and having abiological activity. Herein, the biological activity representativelyrefers to an activity of SHH.

Examples of the factors of the Hedgehog pathway include SHH, PTCH1, GLI1and GLI2.

As used herein, “PTCH1” is one of the factors of the Hedgehog pathway,is a receptor called Patched-1, and is a receptor to which SHH binds. Agene encoding this factor is shown by ptch1. It is stated that, bybinding between SHH and PTCH1, suppression of Smoothened (SMO) which isalso a component of a SHH receptor complex is cancelled, a signal istransmitted into a cell, and finally, transcription of a variety oftarget genes is activated through a Gli transcription factor to exert aphysiological function. It is stated that the gamete mutation of PTCH1causes a hereditary disease of nevoid basal cell carcinoma syndrome(NBCCS) (also called Gorlin syndrome) characterized by minor anomaly andhigh oncogenesis. PTCH1 is also a cancer suppressing gene. This gene isalso known as PTC; BCNS; HPE7; PTC1; PTCH; NBCCS; or PTCH11. Forexample, the factor in human is NC_000009.11 (NCBI Reference Sequence),and NC_000009 (Accession No.) and NM_001083602.1 (Accession No.) (SEQ IDNos.: 43 and 44) are known.

A representative nucleotide sequence of ptch1 can be:

(a) a polynucleotide having a nucleotide sequence of SEQ ID No.: 43<NM_001083602.1> or a fragment sequence thereof;

(b) a polynucleotide encoding a polypeptide consisting of an amino acidsequence of SEQ ID No.: 44 or a fragment thereof;

(c) a polynucleotide encoding a variant polypeptide in which one or moreamino acids have one mutation selected from the group consisting ofsubstitution, addition and deletion in an amino acid sequence of SEQ IDNo.: 44, or a fragment thereof, the variant polypeptide having abiological activity;

(d) a polynucleotide which is a splicing mutant or an allele mutant of anucleotide sequence of SEQ ID No.: 43 or a fragment thereof;

(e) a polynucleotide encoding a species homolog of a polypeptideconsisting of an amino acid sequence of SEQ ID No.: 44 or a fragmentthereof;

(f) a polynucleotide encoding a polypeptide wherein the polynucleotidehybridizes with the polynucleotide of any one of (a) to (e) under astringent condition, and the polypeptide has a biological activity; or

(g) a polynucleotide encoding a polypeptide wherein the polynucleotideconsists of a nucleotide sequence in which the identity to thepolynucleotide of any one of (a) to (e) or a complementary sequencethereof is at least 70%, and the polypeptide has a biological activity.Herein, the biological activity representatively refers to an activityof PTCH1.

An amino acid sequence of PTCH1 can be:

(a) a polypeptide consisting of an amino acid sequence of SEQ ID No.: 44or a fragment thereof;

(b) a polypeptide in which one or more amino acids have one mutationselected from the group consisting of substitution, addition anddeletion in an amino acid sequence of SEQ ID No.: 44, and thepolypeptide has a biological activity;

(c) a polypeptide encoded by a splicing mutant or an allele mutant of anucleotide sequence of SEQ ID No.: 43;

(d) a polypeptide which is a species homolog of an amino acid sequenceof SEQ ID No.: 44; or

(e) a polypeptide having an amino acid sequence in which the identity tothe polypeptide of any one of (a) to (d) is at least 70%, and having abiological activity. Herein, the biological activity representativelyrefers to an activity of PTCH1.

As used herein, “GLI1” is a factor of the Hedgehog (HH) pathway, and oneof transcription factor Gli family members (GLI1, GLI2, GLI3 etc.). Agene encoding GLI1 is shown by gli1. It is stated that GLI1 is locateddownstream from PTCH1, and a signal is transmitted to GPR49/LGR5 fromthis point. It is stated that, by binding between SHH and PTCH1,suppression of Smoothened (SMO) which is also a component of a SHHreceptor complex is cancelled, a signal is transmitted into a cell, andfinally, transcription of a variety of target genes is activated througha GLI transcription factor to exert a physiological function. Forexample, as the factors in human, NM_005269.2 (NCBI Reference Sequence),NM_005269 (Genbank Accession) for variant 1; NM_001160045.1 (NCBIReference Sequence), NM_001160045 (Genbank Accession) for variant 2;NM_001167609.1 (NCBI Reference Sequence), NM_001167609 (GenbankAccession) (SEQ ID Nos.: 45 and 46) for variant 3 are known.

A representative nucleotide sequence of gli1 can be:

(a) a polynucleotide having a nucleotide sequence of SEQ ID No.: 45<NM_001167609.1> or a fragment sequence thereof;

(b) a polynucleotide encoding a polypeptide consisting of an amino acidsequence of SEQ ID No.: 46 or a fragment thereof;

(c) a polynucleotide encoding a variant polypeptide in which one or moreamino acids have one mutation selected from the group consisting ofsubstitution, addition and deletion in an amino acid sequence of SEQ IDNo.: 46, or a fragment thereof, the variant polypeptide having abiological activity;

(d) a polynucleotide which is a splicing mutant or an allele mutant of anucleotide sequence of SEQ ID No.: 45 or a fragment thereof;

(e) a polynucleotide encoding a species homolog of a polypeptideconsisting of an amino acid sequence of SEQ ID No.: 46 or a fragmentthereof;

(f) a polynucleotide encoding a polypeptide wherein the polynucleotidehybridizes with the polynucleotide of any one of (a) to (e) under astringent condition, and the polypeptide has a biological activity; or

(g) a polynucleotide encoding a polypeptide wherein the polynucleotideconsists of a nucleotide sequence in which the identity to thepolynucleotide of any one of (a) to (e) or a complementary sequencethereof is at least 70%, and the polypeptide has a biological activity.Herein, the biological activity representatively refers to an activityof GLI1.

An amino acid sequence of GLI1 can be:

(a) a polypeptide consisting of an amino acid sequence of SEQ ID No.: 46or a fragment thereof;

(b) a polypeptide in which one or more amino acids have one mutationselected from the group consisting of substitution, addition anddeletion in an amino acid sequence of SEQ ID No.: 46, and thepolypeptide has a biological activity;

(c) a polypeptide encoded by a splicing mutant or an allele mutant of anucleotide sequence of SEQ ID No.: 45;

(d) a polypeptide which is a species homolog of an amino acid sequenceof SEQ ID No.: 46; or

(e) a polypeptide having an amino acid sequence in which the identity tothe polypeptide of any one of (a) to (d) is at least 70%, and having abiological activity. Herein, the biological activity representativelyrefers to an activity of GLI1.

As used herein, “GLI2” is a factor of the Hedgehog (HH) pathway, and oneof transcription factor GLI family members (GLI1, GLI2, GLI3 etc.). Agene encoding GLI2 is shown by gli2. It is stated that GLI2 is locateddownstream from PTCH1, and a signal is transmitted to GPR49/LGR5 fromthis point. It is stated that, by binding between Shh and PTCH1,suppression of Smoothened (SMO) which is also a component of a Shhreceptor complex is cancelled, a signal is transmitted into a cell, andfinally, transcription of a variety of target genes is activated througha Gli transcription factor to exert a physiological function. Forexample, NM_005270.4 (NCBI Reference Sequence), NM_005270 (GenbankAccession) (SEQ ID Nos.: 47 and 48) are known as the factors in human.

A representative nucleotide sequence of gli2 can be:

(a) a polynucleotide having a nucleotide sequence of SEQ ID No.: 47<NM_005270.4> or a fragment thereof;

(b) a polynucleotide encoding a polypeptide consisting of an amino acidsequence of SEQ ID No.: 48 or a fragment thereof;

(c) a polynucleotide encoding a variant polypeptide in which one or moreamino acids have one mutation selected from the group consisting ofsubstitution, addition and deletion in an amino acid sequence of SEQ IDNo.: 48, or a fragment thereof, the variant polypeptide having abiological activity;

(d) a polynucleotide which is a splicing mutant or an allele mutant of anucleotide sequence of SEQ ID No.: 47 or a fragment thereof;

(e) a polynucleotide encoding a species homolog of a polypeptideconsisting of an amino acid sequence of SEQ ID No.: 48 or a fragmentthereof;

(f) a polynucleotide encoding a polypeptide wherein the polynucleotidehybridizes with the polynucleotide of any one of (a) to (e) under astringent condition, and the polypeptide has a biological activity; or

(g) a polynucleotide encoding a polypeptide wherein the polynucleotideconsists of a nucleotide sequence in which the identity to thepolynucleotide of any one of (a) to (e) or a complementary sequencethereof is at least 70%, and the polypeptide has a biological activity.Herein, the biological activity representatively refers to an activityof GLI2.

An amino acid sequence of GLI2 can be:

(a) a polypeptide consisting of an amino acid sequence of SEQ ID No.: 48or a fragment thereof;

(b) a polypeptide in which one or more amino acids have one mutationselected from the group consisting of substitution, addition anddeletion in an amino acid sequence of SEQ ID No.: 48, and thepolypeptide has a biological activity;

(c) a polypeptide encoded by a splicing mutant or an allele mutant of anucleotide sequence of SEQ ID No.: 47;

(d) a polypeptide which is a species homolog of an amino acid sequenceof SEQ ID No.: 48; or

(e) a polypeptide having an amino acid sequence in which the identity tothe polypeptide of any one of (a) to (d) is at least 70%, and having abiological activity. Herein, the biological activity representativelyrefers to an activity of GLI2.

Examples of the factors of the Wnt pathway include LRP6 and β-catenin.

As used herein, “LRP6” is one of the factors of the Wnt pathway, and isabbreviation of low-density lipoprotein receptor-related protein 6. Agene encoding this is expressed by lrp6. Gβγ which is G proteinactivates GSK3, and promotes the transcription activity of β-cateninthrough LRP6. For example, as the factors in human, NM_002336.2 (NCBIReference Sequence), NM_002336 (Genbank Accession) LRP6, NM_002336.2(SEQ ID Nos.: 49 and 50) are known.

A representative nucleotide sequence of lrp6 can be:

(a) a polynucleotide having a nucleotide sequence of SEQ ID No.: 49<NM_002336.2> or a fragment thereof;

(b) a polynucleotide encoding a polypeptide consisting of an amino acidsequence of SEQ ID No.: 50 or a fragment thereof;

(c) a polynucleotide encoding a variant polypeptide in which one or moreamino acids have one mutation selected from the group consisting ofsubstitution, addition and deletion in an amino acid sequence of SEQ IDNo.: 50, or a fragment thereof, the variant polypeptide having abiological activity;

(d) a polynucleotide which is a splicing mutant or an allele mutant of anucleotide sequence of SEQ ID No.: 49 or a fragment thereof;

(e) a polynucleotide encoding a species homolog of a polypeptideconsisting of an amino acid sequence of SEQ ID No.: 50 or a fragmentthereof;

(f) a polynucleotide encoding a polypeptide wherein the polynucleotidehybridizes with the polynucleotide of any one of (a) to (e) under astringent condition, and the polypeptide has a biological activity; or

(g) a polynucleotide encoding a polypeptide wherein the polynucleotideconsists of a nucleotide sequence in which the identity to thepolynucleotide of any one of (a) to (e) or a complementary sequencethereof is at least 70%, and the polypeptide has a biological activity.Herein, the biological activity representatively refers to an activityof LRP6.

An amino acid sequence of LRP6 can be:

(a) a polypeptide consisting of an amino acid sequence of SEQ ID No.: 50or a fragment thereof;

(b) a polypeptide in which one or more amino acids have one mutationselected from the group consisting of substitution, addition anddeletion in an amino acid sequence of SEQ ID No.: 50, and thepolypeptide has a biological activity;

(c) a polypeptide encoded by a splicing mutant or an allele mutant of anucleotide sequence of SEQ ID No.: 49;

(d) a polypeptide which is a species homolog of an amino acid sequenceof SEQ ID No.: 50; or

(e) a polypeptide having an amino acid sequence in which the identity tothe polypeptide of any one of (a) to (d) is at least 70%, and having abiological activity. Herein, the biological activity representativelyrefers to an activity of LRP6.

As used herein, “β-catenin” is one of the factors of the Wnt pathway,and a gene encoding this is shown by beta-catenin/CTNNB 1. TheWnt/β-catenin pathway regulates the determination of the destiny of acell in development in a vertebrate and an invertebrate. A Wnt-ligand isa secreted glycoprotein, and binds to a Frizzled receptor. This bindinginitiates a cascade such that GSK-3β as a multifunctional kinase iseliminated from an APC/Axin/GSK-3β complex. It is stated that, whenthere is no Wnt-signal, β-catenin, which is a coupling factor oftranscription, and at the same time, which is embedded in a cellmembrane and exists as an adaptor protein in adhesion between cells, aredegraded by an APC/Axin/GSK-3β complex. It is stated that, whenβ-catenin undergoes proper phosphorylation by the cooperative action ofCK1 and GSK-3β, it leads to ubiquitination and degradation with aproteasome through a β-TrCP/SKP complex. When Wnt binds thereto,Dishevelled (Dvl) undergoes phosphorylation and polyubiquitination to beactivated, and this activation in turn keeps GSK-3β away from thedegradation complex. This causes Rac-1-dependent nuclear translocationto guide to a LEF/TCF DNA-binding factor for stabilizing β-catenin,where it is stated to act as a transcription activation factor bysubstitution with a Groucho-HDAC co-repressor. It is stated that theWnt/β-catenin pathway integrates signals from many other pathways suchas retinoic acid, FGF, TGF-β, and BMP, in many different types of cellsand tissues. For example, as the factors in human, NM_001904.3 (NCBIReference Sequence), NM_001904, XM_942045, XM_945648, XM_945650,XM_945651, XM_945652, XM_945653, XM_945654, XM_945655, XM_945657(Genbank Accession) for variant 1; NM_001098209.1 (NCBI ReferenceSequence), NM_001098209, XM_001133660, XM_001133664, XM_001133673,XM_001133675 (Genbank Accession) for variant 2; and NM_001098210.1 (NCBIReference Sequence), NM_001098210 (Genbank Accession), NM_131059.2(CTNNB, Accession No.) (SEQ ID Nos.: 51 and 52) for variant 3 are known.

A representative nucleotide sequence of β-catenin can be:

(a) a polynucleotide having a nucleotide sequence of SEQ ID No.: 51<NM_131059.2> or a fragment thereof;

(b) a polynucleotide encoding a polypeptide consisting of an amino acidsequence of SEQ ID No.: 52 or a fragment thereof;

(c) a polynucleotide encoding a variant polypeptide in which one or moreamino acids have one mutation selected from the group consisting ofsubstitution, addition and deletion in an amino acid sequence of SEQ IDNo.: 52, or a fragment thereof, the variant polypeptide having abiological activity;

(d) a polynucleotide which is a splicing mutant or an allele mutant of anucleotide sequence of SEQ ID No.: 51 or a fragment thereof;

(e) a polynucleotide encoding a species homolog of a polypeptideconsisting of an amino acid sequence of SEQ ID No.: 52 or a fragmentthereof;

(f) a polynucleotide encoding a polypeptide wherein the polynucleotidehybridizes with the polynucleotide of any one of (a) to (e) under astringent condition, and the polypeptide has a biological activity; or

(g) a polynucleotide encoding a polypeptide wherein the polynucleotideconsists of a nucleotide sequence in which the identity to thepolynucleotide of any one of (a) to (e) or a complementary sequencethereof is at least 70%, and the polypeptide has a biological activity.Herein, the biological activity representatively refers to an activityof β-catenin.

An amino acid sequence of β-catenin can be:

(a) a polypeptide consisting of an amino acid of SEQ ID No.: 52 or afragment thereof;

(b) a polypeptide in which one or more amino acids have one mutationselected from the group consisting of substitution, addition anddeletion in an amino acid sequence of SEQ ID No.: 52, and thepolypeptide has a biological activity;

(c) a polypeptide encoded by a splicing mutant or an allele mutant of anucleotide sequence of SEQ ID No.: 51;

(d) a polypeptide which is a species homolog of an amino acid sequenceof SEQ ID No.: 52; or

(e) a polypeptide having an amino acid sequence in which the identity tothe polypeptide of any one of (a) to (d) is at least 70%, and having abiological activity. Herein, the biological activity representativelyrefers to an activity of β-catenin.

As used herein, purmorphamine is another name ofN-(4-morpholinophenyl)-2-(1-naphthyloxy)-9-cyclohexyl-9H-purine-6-amine,and is a known compound having a CAS number of 483367-10-8. It is knownas an agonist of a Frizzled family (membrane protein responsible forHedgehog signal transmission route) of a seven-transmembrane proteincalled Smoothened. Therefore, in the present invention, it can be usedas an agonist of SHH, for example, as an agonist of a Frizzled familysuch as purmorphamine.

As used herein, a “protein”, a “polypeptide”, an “oligopeptide” and a“peptide” are herein used in the same meaning, and refer to a polymer ofamino acids having any length. This polymer may be straight or branched,or cyclic. An amino acid may be natural or non-natural, or may be analtered amino acid. This term can also encompass an assembly of acomplex of a plurality of polypeptide chains. This term also encompassesa natural or artificially altered amino acid polymer. Examples of suchalterations include disulfide bond formation, glycosylation, lipidation,acetylation, phosphorylation and any other manipulation or alteration(e.g., binding with a label component). This definition also encompassesa polypeptide containing one, two or more analogs of amino acids (e.g.,including non-natural amino acid), a peptide-like compound (e.g.,peptoid) and other alterations known in the art.

As used herein, an “amino acid” may be natural or non-natural, as far asthe object of the present invention is satisfied.

As used herein, a “polynucleotide”, an “oligonucleotide” and a “nucleicacid” are herein used in the same meaning, and refer to a polymer ofnucleotides having any length. This term also encompasses an“oligonucleotide derivative” and a “polynucleotide derivative”. The“oligonucleotide derivative” or the “polynucleotide derivative” includesa derivative of a nucleotide, or refers to an oligonucleotide or apolynucleotide in which a bond between nucleotides is different from thenormal bond, and the terms are used interchangeably. Specific examplesof such an oligonucleotide include 2′-O-methyl-ribonucleotide, anoligonucleotide derivative in which a phosphodiester bond in anoligonucleotide is converted into a phosphorothioate bond, anoligonucleotide derivative in which a phosphodiester bond in anoligonucleotide is converted into an N3′-P5′ phosphoramidate bond, anoligonucleotide derivative in which ribose and a phosphodiester bond inan oligonucleotide are converted into a peptide nucleic acid bond, anoligonucleotide derivative in which uracil in an oligonucleotide issubstituted with C-5 propynyluracil, an oligonucleotide derivative inwhich uracil in an oligonucleotide is substituted with C-5thiazoleuracil, an oligonucleotide derivative in which cytosine in anoligonucleotide is substituted with C-5 propynylcytosine, anoligonucleotide derivative in which cytosine in an oligonucleotide issubstituted with phenoxazine-modified cytosine, an oligonucleotidederivative in which ribose in DNA is substituted with 2′-O-propylribose,and an oligonucleotide derivative in which ribose in an oligonucleotideis substituted with 2′-methoxyethoxyribose. Unless otherwise indicated,it is intended that a specified nucleic acid sequence also includes apreservatively altered variant (e.g., degenerate codon substitution) anda complementary sequence thereof, like an explicitly shown sequence.Specifically, a degenerate codon substitution is attained by making asequence in which a third position of one or more selected (or all)codons is substituted with a mixed base and/or a deoxyinosine residue(Batzer et al., Nucleic Acid Res. 19: 5081 (1991); Ohtsuka et al., J.Biol. Chem. 260: 2605-2608 (1985); Rossolini et al., Mol. Cell. Probes8: 91-98 (1994)). As used herein, a “nucleic acid” is usedinterchangeably with a gene, cDNA, mRNA, an oligonucleotide, and apolynucleotide. As used herein, a “nucleotide” may be natural ornon-natural.

As used herein, a “gene” refers to an agent defining a genetic trait.Genes are usually arranged on a chromosome in a certain order. A genedefining a primary structure of a protein is referred to as a structuralgene, and a gene influencing on the expression thereof is referred to asa regulator gene. As used herein, a “gene” may refer to a“polynucleotide”, an “oligonucleotide” and a “nucleic acid”.

As used herein, “homology” between genes refers to a degree of identityof two or more gene sequences to each other, and having “homology”generally refers to a high degree of identity or similarity. Therefore,as the homology between certain two genes is higher, identity orsimilarity between those sequences is higher. Whether two kinds of geneshave homology or not can be examined by direct comparison of sequences,or in the case of a nucleic acid, by a hybridization method under astringent condition. In the case where two gene sequences are directlycompared, those genes have homology when DNA sequences of the genesequences are representatively at least 50% identical between the genesequences, preferably when the DNA sequences are at least 70% identical,more preferably when the DNA sequences are at least 80%, 90%, 95%, 96%,97%, 98% or 99% identical. Therefore, as used herein, a “homolog” or a“homologous gene product” means a protein in another species, preferablya mammal, which exerts the same biological function as that of a proteinconstituent of a complex which will be further described herein. Such ahomolog may also be named as an “ortholog gene product”. The algorithmfor detecting an ortholog gene pair from human, a mammal or otherspecies uses the whole genome of these organisms. First, a pairing besthit is recovered using expected complete Smith-Waterman Alignment ofproteins. In order to further improve reliability, a cluster of thesepairs may be formed using a pairing best hit including Drosophilamelanogaster and C. elegans proteins. Such analysis is provided, forexample, in Nature, 2001, 409: 860-921. Based on sequence homology ongenes of other species of genes encoding proteins provided. As usedherein, homologs of the proteins described herein can be isolated byapplying a conventional technique to clone respective genes, andexpressing proteins from such genes, or by isolating similar complexesaccording to the method provided herein, or according to anotherappropriate method well-known in the art.

An amino acid may be referred herein to by any of the generally knownthree letter symbol, or one letter symbol recommended by the IUPAC-IUBBiochemical Nomenclature Commission. A nucleotide may be similarlyreferred to by the generally recognized one letter code. As used herein,comparison of similarity, identity and homology of amino acid sequencesand nucleotide sequences can be made based on the calculation by usingdefault parameters employing BLAST which is a tool for analyzingsequences. Retrieval of identity can be performed, for example, usingBLAST 2.2.9 of NCBI (published on May 12, 2004). A value of identity asused herein usually refers to a value obtained by alignment under thedefault condition using the BLAST, however, in the case where a highervalue is obtained by change in the parameters, the highest value isadopted as the value of identity. In the case where identity isevaluated in a plurality of regions, among the obtained values, thehighest value is adopted as the value of identity. Similarity is anumerical value obtained by also considering similar amino acids, inaddition to identity.

As used herein, a polynucleotide which “hybridizes under a stringentcondition” refers to a well-known condition which is conventionally usedin the art. Such a polynucleotide can be obtained by performing a colonyhybridization method, a plaque hybridization method or a Southern blothybridization method using a polynucleotide selected from thepolynucleotides of the present invention as a probe. Specifically, sucha polynucleotide means a polynucleotide which can be identified byperforming hybridization at 65° C. in the presence of 0.7 to 1.0 M NaClusing a filter on which DNA derived from a colony or a plaque isimmobilized, and washing the filter under a condition of 65° C. using aSSC (saline-sodium citrate) solution having a 0.1 to 2-foldconcentration (the composition of the SSC solution having a 1-foldconcentration is 150 mM sodium chloride and 15 mM sodium citrate).Hybridization can be performed in accordance with the methods describedin experimental texts such as Molecular Cloning 2nd ed., CurrentProtocols in Molecular Biology, Supplement 1-38, DNA Cloning 1: CoreTechniques, A Practical Approach, Second Edition, Oxford UniversityPress (1995). Herein, a sequence containing only an A sequence or only aT sequence is preferably eliminated from sequences which hybridize undera stringent condition. Therefore, a polypeptide used in the presentinvention (e.g., transthyretin) also includes a polypeptide encoded by anucleic acid molecule which hybridizes with a nucleic acid moleculeencoding a polypeptide particularly described in the present inventionunder a stringent condition. These low stringency conditions includehybridization at 40° C. for 18 to 20 hours in a buffer containing 35%formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02%BSA, 100 μg/ml denatured salmon sperm DNA, and 10% (weight/volume)dextran sulfate, washing the resultant at 55° C. for 1 to 5 hours in abuffer consisting of 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1%SDS, and washing the resultant at 60° C. for 1.5 hours in a bufferconsisting of 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS.

As used herein, a “purified” substance or biological an agent (e.g.,nucleic acid or protein) refers to a substance or a biological an agentfrom which at least a part of an agent naturally associated with thebiological agent has been removed. Therefore, usually, the purity of abiological agent in a purified biological agent is higher than that inthe state where the biological agent usually exists (that is,concentrated). The term “purified” used herein means that preferably atleast 75% by weight, more preferably at least 85% by weight, furtherpreferably at least 95% by weight, and most preferably at least 98% byweight of the same type of a biological agent exists. A substance usedin the present invention is preferably a “purified” substance.

As used herein, a “corresponding” amino acid or nucleic acid refers toan amino acid or a nucleotide which has or is expected to have, in acertain polypeptide molecule or polynucleotide molecule, an actionsimilar to that of a predetermined amino acid or nucleotide in apolypeptide or a polynucleotide as a standard of comparison, andparticularly, in the case of an enzyme molecule, refers to an amino acidwhich exists at a similar position in an active site and makes a similarcontribution to the catalytic activity. For example, in the case of anantisense molecule, it can be a similar part in an orthologcorresponding to a specified part of the antisense molecule. Acorresponding amino acid can be a specified amino acid which issubjected to, for example, cysteination, glutathionylation, S—S bondformation, oxidation (e.g., oxidation of methionine side chain),formylation, acetylation, phosphorylation, glycosylation, myristylationor the like. Alternatively, a corresponding amino acid can be an aminoacid responsible for dimerization. Such a “corresponding” amino acid ornucleic acid may be a region or a domain over a certain range.Therefore, in such a case, as used herein, it is named as a“corresponding” region or domain.

As used herein, a “corresponding” gene (e.g., polynucleotide sequence ormolecule) refers to a gene (e.g., polynucleotide sequence or molecule)of a certain species which has or is expected to have an action similarto that of a predetermined gene in a species being a standard ofcomparison, and when there is a plurality of genes having such anaction, the corresponding gene refers to a gene having evolutionarilythe same origin. Therefore, a gene corresponding to a certain gene canbe an ortholog of the gene. Therefore, regarding RPG49 and R-spondins ofa mouse or a rat, corresponding RPG49 and R-spondins can be found,respectively, in human. Such a corresponding gene can be identifiedusing a technique well-known in the art. Therefore, for example, acorresponding gene in a certain animal (e.g., mouse), or a gene being astandard of a corresponding gene (e.g., RPG49, R-spondins, or shh) canbe found by retrieval from sequence database of the animal (e.g., humanor rat) using sequences such as SEQ ID Nos.: 1, 3, 5, 7, 9, and 11 as aquery sequence.

As used herein, a “fragment” refers to a polypeptide or a polynucleotidehaving a sequence length of 1 to n-1, relative to a full lengthpolypeptide or polynucleotide (length: n). The length of the fragmentcan be appropriately changed depending on the purpose thereof. Examplesof the lower limit of the length, in the case of a polypeptide, include3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50 and more amino acids,and a length which is represented by an integer not specifically listedherein (e.g., 11) can also be proper as the lower limit. In addition, inthe case of a polynucleotide, examples of the lower limit include 5, 6,7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 and more nucleotides, and alength represented by an integer not specifically listed herein (e.g.,11) can also be proper as the lower limit. As used herein, concerningsuch a fragment, it is understood that, for example, when a full lengthpolypeptide or polynucleotide functions as a marker, the fragment itselfis also within the scope of the present invention, as far as it has afunction as a marker.

According to the present invention, the term “activity” as used hereinrefers to the function of a molecule in a broadest sense. The activityis not intended to be particularly limited, but generally includes abiological function, a biochemical function, a physical function or achemical function of a molecule. The activity includes, for example, anenzyme activity, an ability to interact with other molecules, an abilityto activate, promote, stabilize, inhibit, suppress, or destabilize thefunction of other molecules, the stability, and an ability to belocalized at a specified position in a cell. When applicable, this termalso relates to the function of a protein complex in a broadest sense.

As used herein, the “biological function” used when referring to acertain gene or a nucleic acid molecule or a polypeptide relatingthereto, refers to a specified function which can be possessed by thegene, the nucleic acid molecule or the polypeptide in a living body.This function includes, but is not limited to, production of a specificantibody, the enzyme activity, and impartation of resistance. In thepresent invention, this function includes, but is not limited to, afunction of GPR49/LGR5 or the like of recognizing an R-spondin. As usedherein, the biological function can be exerted by the “biologicalactivity”. As used herein, the “biological activity” refers to theactivity which can be possessed by a certain agent (e.g., polynucleotideor protein) in a living body, includes an activity of exerting a varietyof functions (e.g., transcription promoting activity), and for example,includes an activity of activating or inactivating another molecule byinteraction with a certain molecule. When two agents interact, thebiological activity is binding between two molecules and biologicalchange caused by the binding, for example, when one molecule is settledusing an antibody, the other molecule is also settled, and the twomolecules are thought to be bound together. Therefore, observation ofsuch coprecipitation is one determination procedure. For example, when acertain agent is an enzyme, the biological activity includes the enzymeactivity. In another example, when a certain agent is a ligand, thebiological activity includes binding of the ligand to a correspondingreceptor. Such a biological activity can be measured by a techniquewell-known in the art. Therefore, the “activity” refers to a variety ofmeasurable indices which show or reveal a bond (either direct orindirect); and influence on a response (that is, having a measurableinfluence in response to some exposure or stimulation), and examplesthereof include affinity of a compound which directly binds to thepolypeptide or the polynucleotide of the present invention, the amountof proteins upstream or downstream after some stimulations or events, orthe measure of other similar functions.

As used herein, “expression” of a gene, a polynucleotide, a polypeptideor the like refers to that the gene or the like receives a certainaction in vivo to be converted into another form. Preferably, theexpression refers to that a gene, a polynucleotide or the like istranscribed and translated into the form of a polypeptide, andtranscription to make mRNA can also be one aspect of the expression.More preferably, such a form of a polypeptide can be a form which hasundergone processing after translation (derivative referred to herein).For example, the expression level of GPR49/LGR5 can be determined by anymethod. Specifically, by evaluating the amount of mRNA of GPR49/LGR5,the amount of a GPR49/LGR5 protein, and the biological activity of aGPR49/LGR5 protein, the expression level of GPR49/LGR5 can be known. Theamount of mRNA or a protein of GPR49/LGR5 can be determined by themethod described herein.

As used herein, a “functional equivalent” refers to any matter havingthe same objective function but a different structure relative to theoriginal entity as a subject. Therefore, when referring to “R-spondinsor a functional equivalent thereof” or “the group consisting ofR-spondins and a functional equivalent thereof”, it is understood that afunctional equivalent includes, in addition to R-spondins themselves, amutant or a variant of R-spondins (e.g., amino acid sequence variant)which has an action of controlling differentiation and/or promotingproliferation of an eye cell or the like, as well as one which can bechanged into R-spondins themselves, or a mutant or a variant of theR-spondins (e.g., including nucleic acids encoding R-spondins themselvesor a mutant or a variant of R-spondins, and a vector, a cell or the likecomprising the nucleic acid) at the time point of the action. In thepresent invention, it is understood that a functional equivalent of anR-spondin can be used similarly to an R-spondin, unless particularlyreferred to.

In addition, when referring to “GPR49/LGR5 or a functional equivalentthereof” or “the group consisting of GPR49/LGR5 and a functionalequivalent thereof”, it is understood that they include, in addition toGPR49/LGR5 itself, a mutant or a variant (e.g., amino acid sequencevariant) of GPR49/LGR5 which has an action of controllingdifferentiation and/or promoting proliferation of an eye cell or thelike, or has a function as the marker described herein, as well as onewhich can be changed to GPR49/LGR5 itself or a mutant or a variant ofGPR49/LGR5 (e.g., including a nucleic acid encoding GPR49/LGR5 itself ora mutant or a variant of GPR49/LGR5, and a vector, a cell or the likecomprising the nucleic acid) at the time point of the action. In thepresent invention, it is understood that a functional equivalent ofGPR49/LGR5 can be used similarly to GPR49/LGR5, unless particularlyreferred to.

In addition, when referring to “SONIC HEDGEHOG (SHH) or a functionalequivalent thereof” or “the group consisting of SONIC HEDGEHOG (SHH) anda functional equivalent thereof”, it is understood that they include, inaddition to SHH itself, a mutant or a variant (e.g., amino acid sequencevariant) of SHH which has an action of controlling differentiationand/or promoting proliferation of an eye cell or the like, or has afunction as the marker described herein, as well as one which can bechanged to SHH itself or a mutant or a variant of SHH (e.g., including anucleic acid encoding SHH itself or a mutant or a variant of SHH, and avector, a cell or the like comprising the nucleic acid) at the timepoint of the action. In the present invention, it is understood that afunctional equivalent of SHH can be used similarly to SHH, unlessparticularly referred to.

When referring to “an agent suppressing GPR49/LGR5 or a functionalequivalent thereof” or “the group consisting of an agent suppressingGPR49/LGR5 and a functional equivalent thereof”, it is understood thatthey include, in addition to an agent suppressing GPR49/LGR5 itself, amutant or a variant (e.g., synthetic variant) of an agent suppressingGPR49/LGR5 which is changed so as to have an action of controllingdifferentiation and/or promoting proliferation of an eye cell or thelike (can be changed to an agent suppressing GPR49/LGR5 (e.g., aprecursor or a prodrug such as an ester)) at the time point of theaction. In the present invention, it is understood that a functionalequivalent of the agent suppressing GPR49/LGR5 can be used similarly tothe agent suppressing GPR49/LGR5, unless particularly referred to.

In addition, when referring to “an agonist of SHH (e.g., agonist ofFrizzled family such as purmorphamine) or a functional equivalentthereof” or “the group consisting of an agonist of SHH (e.g., agonist ofFrizzled family such as purmorphamine) and a functional equivalentthereof”, it is understood that they include, in addition to an agonistof SHH (e.g., agonist of Frizzled family such as purmorphamine), amutant or a variant (e.g., synthetic variant) of an agonist of SHH(e.g., agonist of Frizzled family such as purmorphamine) which ischanged so as to have an action of controlling differentiation and/orpromoting proliferation of an eye cell or the like (and is changed to anagonist of SHH (e.g., agonist of Frizzled family such as purmorphamine)itself or a mutant or a variant of an agonist of SHH (e.g., agonist ofFrizzled family such as purmorphamine) (e.g., a precursor or a prodrugsuch as an ester)) at the time point of the action. In the presentinvention, it is understood that a functional equivalent of an agonistof SHH (e.g., agonist of Frizzled family such as purmorphamine) can beused similarly to the agonist of SHH (e.g., agonist of Frizzled familysuch as purmorphamine), unless particularly referred to.

As used herein, an “agonist” refers to a substance which makes an entityas a subject (e.g., receptor) expresses the biological action of thereceptor. Examples thereof include a synthesized agonist and an alteredagonist, in addition to a natural agonist (also named as a ligand).

As used herein, an “antagonist” refers to a substance which, relative toan entity (e.g., receptor) as a subject, inhibits the biological actionof the receptor. There are an antagonist which inhibits the actionnon-competitively with an agonist (or a ligand), in addition to anantagonist which inhibits the action competitively with an agonist (or aligand). An antagonist can also be obtained by altering an agonist.Since an antagonist inhibits a physiological phenomenon, it can beincluded in the concept of an inhibitor or a suppressing (suppressing)agent.

As used herein, an “agent inhibiting (GPR49/LGR5 etc.)” refers to anagent which can reduce or eliminate the function of GPR49/LGR5 or thelike as a subject temporarily or permanently. Examples of such an agentinclude, but are not limited to, an antibody, an antigen-bindingfragment thereof, a derivative thereof, and agents in the form of anucleic acid such as antisense and RNAi agent such as siRNA.

A functional equivalent of GPR49/LGR5 and R-spondins used in the presentinvention can be found by retrieval. from database and the like. As usedherein, “retrieval” refers to finding out other nucleic acid nucleotidesequences a having specified function and/or nature utilizing a certainnucleic acid nucleotide sequence electronically or by a biological orother method. Examples of electronic retrieval include, but are notlimited to BLAST (Altschul et al., J. Mol. Biol. 215: 403-410 (1990)),FASTA (Pearson & Lipman, Proc. Natl. Acad. Sci., USA 85: 2444-2448(1988)), Smith and Waterman method (Smith and Waterman, J. Mol. Biol.147: 195-197 (1981)), and Needleman and Wunsch method (Needleman andWunsch, J. Mol. Biol. 48: 443-453 (1970)). Examples of biologicalretrieval include, but are not limited to stringent hybridization, amicroarray in which genome DNA is stuck on a nylon membrane or the like,or a microarray stuck to a glass plate (microarray assay), PCR and insitu hybridization. As used herein, it is intended that a gene used inthe present invention should include a corresponding gene identified bysuch electronic retrieval or biological retrieval.

As the functional equivalent of the present invention, a functionalequivalent in which, in an amino acid sequence, one or a plurality ofamino acids are inserted, substituted or deleted, or added to one orboth ends thereof can he used. As used herein, “in an amino acidsequence, one or a plurality of amino acids are inserted, substituted ordeleted, or added to one or both ends thereof” means that the alterationhas been performed by a well-known technical method such assite-specific metagenesis, or by substitution of a plurality of aminoacids by natural mutation to the extent that they are generatednaturally.

An altered amino acid sequence of GPR49/LGR5, R-spondins, the factors ofthe Hedgehog pathway such as SHH, PTCH1, GLI1, and GLI2, the factors ofthe Wnt pathway such as LRP6 and β-catenin, and the like can be the onein which, for example, insertion, substitution, or deletion, or additionto one or both ends of 1 to 30, preferably 1 to 20, more preferably 1 to9, further preferably 1 to 5, particularly preferably 1 to 2 amino acidsis performed. An altered amino acid sequence is preferably a sequence inwhich an amino acid sequence thereof may be an amino acid sequence inwhich an amino acid sequence of GPR49/LGR5, R-spondins, the factors ofthe Hedgehog pathway such as SHH, PTCH1, GLI1, and GLI2, the factors ofthe Wnt pathway such as LRP6 and β-catenin, or the like has 1 or aplurality of (preferably, 1 or a few, or 1, 2, 3 or 4) preservativesubstitutions. Herein, the “preservative substitution” means that 1 or aplurality of amino acid residues are substituted with another chemicallysimilar amino acid residue so that the function of a protein is notsubstantially altered. For example, there are the case where a certainhydrophobic residue is substituted with another hydrophobic residue, thecase where a certain polar residue is substituted with another polarresidue having the same charge, and the like. A functionally similaramino acid which can be subjected to such substitution is known in theart for every amino acid. Specific examples thereof include, as anon-polar (hydrophobic) amino acid, alanine, valine, isoleucine,leucine, proline, tryptophan, phenylalanine, and methionine. Examples ofa polar (neutral) amino acid include glycine, serine, threonine,tyrosine, glutamine, asparagine, and cysteine. Examples of a (basic)amino acid having positive charge include arginine, histidine, andlysine. In addition, examples of an (acidic) amino acid having negativecharge include aspartic acid and glutamic acid.

As used herein, a “marker (substance or gene)” refers to a substancewhich serves as a criterion for pursuing whether a subject is in acertain state (e.g., the level, or the presence or absence of a diseasestate, a disorder state, a proliferation ability, or a differentiatedstate) or not, or whether there is such a risk or not. Examples of sucha marker include a gene, a gene product, a metabolite, and an enzyme. Inthe present invention, detection, diagnosis, preliminary detection,prediction or advance diagnosis concerning the certain state (e.g., adisease such as differentiation disorder) can be realized using apharmaceutical, a drug, an agent, a factor or means specific for amarker associated with the state, or a composition, a kit, a system ofthe like comprising them. As used herein, a “gene product” refers to aprotein encoded by a gene or mRNA. As used herein, it was found that agene product for which association with an eye cell has not been shown(i.e. GPR49/LGR5, etc.) can be used as an index of differentiation of aneye cell.

As used herein, a “nerve cell” is used in a broad sense, and means anycell included in an organ of a nervous system. Particularly, it isunderstood that a cell derived from a neural crest cell (e.g., cornealendothelial cell) is also included.

As used herein, an “eye cell” is used in a broad sense, and means anycell existing in an eye. The eye cell includes any cell existing ineyelid, sclera, cornea, uvea, crystalline lens, vitreous body, retina,and optic nerve.

As used herein, a “corneal endothelial cell” is used in the ordinarymeaning used in the art. The cornea is one of lamellar tissuesconstituting an eye, is transparent, and is positioned at a part closestto the outside world. In human, the cornea is stated to be composed offive layers from the external side (body surface) in order, and iscomposed of corneal epithelium, Bowman's membrane (external boundaryline), Lamina propria, Descemet's membrane (internal boundary line), andcorneal endothelium from the external side. Unless specified, partsother than epithelium and endothelium may be collectively named as“corneal stroma”, and are named so used herein.

As used herein, a “corneal tissue” is used in an ordinary sense, andrefers to a tissue itself constituting the cornea. When referring to acorneal tissue, it may include all of corneal epithelium, Bowman'smembrane (external boundary line), Lamina propria, Descemet's membrane(internal boundary line), and corneal endothelium (in the case of human;in the case of other animals, all of the above as appropriate dependingon the classification corresponding thereto), or may lack a part, or mayinclude another tissue (sclera) in addition to the cornea. In such acase, it may be particularly called sclerocornea. Therefore, it ispossible to state that the sclerocornea or a sclerocornea slice is oneembodiment of a corneal tissue.

As used herein, a “proliferation ability” refers to an ability of a cellto proliferate. As used herein, unless particularly referred to, thestate of proliferation shows a possibility of proliferation in astationary state. Herein, the “stationary state” refers to a state wherehomeostasis of a living body is maintained under the normal condition ofthe living body. Such a state can be easily determined by a personskilled in the art. For example, such a state can be confirmed byanalysis of the cell density based on that the cell density isapproximately constant and does not change, or that expression of a cellproliferation marker is not recognized.

As used herein, a “high proliferation ability” refers to that a cell hasthe proliferation ability in the stationary state.

As used herein, “proliferation promotion” refers to that theproliferation state of a certain cell is promoted. In the case where asubject cell has not proliferated, when proliferation is initiated ifonly a little, this corresponds to proliferation promotion, and in thecase of a cell which has been proliferating already, when theproliferation level is maintained or enhanced, preferably, enhanced,this corresponds to proliferation promotion.

As used herein, a “differentiation ability” refers to the ability of acell to differentiate. It can be said that a cell having thedifferentiation ability is “undifferentiated” in that sense. Since astem cell (embryonic stem cell, reproduction cell, iPS cell, tissue stemcell, etc.) can be further differentiated, it can be said as having thedifferentiation ability.

As used herein, an “undifferentiated cell” refers to any cell having thedifferentiation ability. Therefore, the undifferentiated cell includes acell which has differentiated to a certain extent, but can stilldifferentiate, in addition to a stem cell.

As used herein, “differentiation suppression” refers to suppression ofdifferentiation. It is understood that both of the case where thedifferentiation level is unchanged if a cell is not differentiated, andthe case where the differentiation level is advancing toward anundifferentiation direction are included in the “differentiationsuppression”.

As used herein, an “agent for suppressing differentiation and/orpromoting proliferation”, when referring to a substance or an agent,refers to an agent which can suppress differentiation of the cell as asubject and/or promote proliferation of the cell. As used herein,particularly, the “agent for suppressing differentiation and/orpromoting proliferation” includes at least one kind selected from thegroup consisting of R-spondins, SHH, an agonist of SHH (e.g., agonist ofFrizzled family such as purmorphamine), an agent suppressing GPR49/LGR5(antagonist) and a functional equivalent thereof.

As used herein, a “stem cell” refers to a cell having both of theability to differentiate into cells of a plurality of lineages(pluripotency), and the ability to maintain pluripotency even after celldivision (ability to self-renew). The stem cell includes an embryonicstem cell, a reproduction cell, an iPS cell, and a tissue stem cell.

As used herein, a “specimen” refers to a subject which is to besubjected to diagnosis or detection in the present invention (e.g., anorganism such as human or an organ (eye) or a cell which has been takenout from an organism).

As used herein, a “sample” refers to any substance obtained from aspecimen or the like, and includes, for example, a cell of an eye. Aperson skilled in the art can appropriately select a preferable samplebased on the descriptions described herein.

As used herein, a “colony forming ability”, when referring to that of acell, refers to an ability to form a colony. As used herein, the colonyforming ability can be determined using, for example, a colony formationtest of a cell under culturing conditions known in the art, as astandard test method.

As used herein, “Ki-67” is a cell proliferation marker, and isrepresentatively a cell cycle-associated nucleoprotein represented bythe accession number P46013. In a cell during proliferation, Ki-67 isexpressed in the G₁ phase, the S phase, the G₂ phase and the M phase,but Ki-67 does not exist in the G₀ phase in which proliferation pauses,and therefore, it is used as a marker of cell proliferation and cellcycle. In addition, since a positive correlation is observed between theKi-67 expression quantity and malignancy of a tumor, Ki-67 is alsouseful as a marker for detecting a proliferating cell in a tumor tissue.

As used herein, “Ki-67 positivity” refers to that Ki-67 being a cellmarker is expressed in a target cell.

As used herein, “BrdU” is abbreviation of brominated deoxyuridine. SinceBrdU is taken as an analog of dTTP during DNA synthesis, DNA (cellnucleus) which has taken BrdU can be detected with an antibody specificfor BrdU which has been taken into DNA.

As used herein, “BrdU positivity” refers to that BrdU being a cellmarker is expressed in a target cell.

As used herein, an “agent” is used in a broad sense, and may be anysubstance or other elements (e.g., energy such as light, radioactivity,heat, and electricity), as far as an intended object can be attained.Examples of such a substance include, but are not limited to a protein,a polypeptide, an oligopeptide, a peptide, a polynucleotide, anoligonucleotide, a nucleotide, a nucleic acid (e.g., including DNA suchas cDNA and genome DNA, and RNAs such as mRNA), a polysaccharide, anoligosaccharide, a fat, an organic low molecule (e.g., a hormone, aligand, an information transmitting substance, an organic low molecule,a molecule synthesized by combinatorial chemistry, and a low moleculewhich can be utilized as a medicine (e.g., a low-molecular weightligand)), and a composite molecule thereof. Representative examples ofan agent specific for a polynucleotide include, but are not limited to apolynucleotide having complementarity by certain sequence homology(e.g., 70% or more sequence identity) relative to a sequence of thepolynucleotide, and a polypeptide such as a transcription factor bindingto a promoter region. Representative examples of an agent specific for apolypeptide include, but are not limited to an antibody specificallydirected to the polypeptide or a derivative thereof or an analog thereof(e.g., single-stranded antibody), a specific ligand or receptor when thepolypeptide is a receptor or a ligand, and, when the polypeptide is anenzyme, a substrate.

As used herein, a “detection agent” in a broad sense refers to any drugby which an objective subject can be detected.

As used herein, a “diagnostic agent (or diagnostic)” in a broad senserefers to any agent by which an objective condition (e.g., a disease)can be diagnosed.

As used herein, a “therapeutic agent (or therapeutic)” in a broad senserefers to any agent by which an objective condition (e.g., a disease)can be treated.

As used herein, a “preventive agent (or preventive)” in a broad senserefers to any agent by which an objective condition (e.g., a disease)can be prevented. As used herein, a “progression preventive agent(progression preventive)”, regarding a certain disease or the like,refers to any agent by which progression of the condition can beprevented.

As used herein, “in vivo” refers to the inside of a living body. In aspecific context, “inside a living body” refers to a position at whichan objective substance should he arranged.

As used herein, “in vitro” refers to a state where a part of a livingbody is isolated or liberated “outside a living body” (e.g., into a testtube) for a variety of research objectives. This is a term in contrastto in vivo.

As used herein, “ex vivo” refers to a series of motions, in the casewhere certain treatment is performed outside a body, and thereafter, apart of the body is intended to be returned to the inside of the body.Also in the present invention, it is possible to simulate an embodimentin which a cell in a living body is treated with an agent of the presentinvention, and is returned to a patient again.

(Preferable Embodiments)

Preferable embodiments of the present invention will be illustratedbelow. Embodiments provided below are provided for better understandingof the present invention, and it is understood that the scope of thepresent invention should not be limited to the following descriptions.Therefore, it is clear that a person skilled in the art canappropriately perform the alteration within the scope of the presentinvention, in view of the descriptions described herein.

(Differentiation Suppression and/or Proliferation Promotion)

In one aspect, the present invention provides an agent for suppressingdifferentiation and/or promoting proliferation of a cell, comprising atleast one kind selected from the group consisting of R-spondins, SHH, anagonist of SHH (e.g., agonist of Frizzled family such as purmorphamine),an agent suppressing GPR49/LGR5 and a functional equivalent thereof.Examples of the cell which is a subject of the present invention mayinclude, but are not particularly limited to, an eye cell, a nerve cellincluding a cell derived from a neural crest cell (including a cornealendothelial cell), and epithelial cells such as a conjunctivalepithelial cell, an amniotic epithelial cell, an oral mucosa epithelialcell, a nose mucosa epithelial cell, and a corneal epithelial cell. In apreferable embodiment, a cell which is a subject of the presentinvention is an eye cell. The eye cell which is a subject of the presentinvention can include, but is not limited to a retinal cell, a vitreousbody cell, a corneal epithelial cell, a corneal parenchymal cell and acorneal endothelial cell. A functional equivalent of R-spondins includesa mutant or a variant of R-spondins (e.g., amino acid sequence variant)which has an action of controlling differentiation and/or promotingproliferation of an eye cell or the like, and as well as one which canbe changed to R-spondins themselves or a mutant or a variant ofR-spondins (e.g., including a nucleic acid encoding R-spondinsthemselves or a mutant or a variant of R-spondins, and a vector, a cellor the like comprising the nucleic acid) at the time point of theaction. A functional equivalent of SHH includes a mutant or a variant ofSHH (e.g., amino acid sequence variant) which has an action ofcontrolling differentiation and/or promoting proliferation of an eyecell or the like, or has a function as the marker described herein, aswell as one which can be changed to SHH itself or a mutant or a variantof SHH (e.g., including a nucleic acid encoding SHH itself or a mutantor a variant of SHH, and a vector, a cell or the like comprising thenucleic acid) at the time point of the action. A functional equivalentof an agonist of SHH (e.g., agonist of Frizzled family such aspurmorphamine) includes a mutant or a variant (e.g., synthetic variant)of an agonist of SHH (e.g., agonist of Frizzled family such aspurmorphamine) which has an action of controlling differentiation and/orpromoting proliferation of an eye cell or the like, or has a function asthe marker herein described as well as one which can be changed into anagonist of SHH (e.g., agonist of Frizzled family such as purmorphamine)itself or a mutant or a variant of this agonist of SHH (e.g., agonist ofFrizzled family such as purmorphamine) (e.g., a precursor or a prodrugsuch as an ester) at the time point of the action. A functionalequivalent of an agent suppressing GPR49/LGR5 includes a mutant or avariant (e.g., synthetic variant) of an agent suppressing GPR49/LGR5which is changed so as to have an action of controlling differentiationand/or promoting proliferation of an eye cell or the like (can bechanged into an agent suppressing GPR49/LGR5 (e.g., a precursor or aprodrug such as an ester)) at the time point of the action.

In one embodiment, R-spondins used in the present invention include atleast one selected from R-spondin 1, R-spondin 2, R-spondin 3 andR-spondin 4.

In a preferable embodiment, R-spondins used in the present inventioninclude R-spondin 1.

In one embodiment, an eye cell which is a subject of the presentinvention is a cell which does not proliferate in the stationary state.Not wishing to be bound by any theory, the present invention exerts aneffect which has not been obtained previously in a point that even acell which does not proliferate in the stationary state can beproliferated by the present invention.

In another embodiment, an eye cell which is a subject of the presentinvention includes at least one kind of cell selected from a retinalcell, a vitreous body cell, a corneal epithelial cell, a cornealparenchymal cell and a corneal endothelial cell. Particularly, a cornealendothelial cell is a cell which proliferates little in the stationarystate, and a point that this cell can be proliferated has an extremelyimportant meaning from the viewpoint of treatment or prevention of anophthalmological disease. Therefore, in one preferable embodiment, aneye cell which is a subject of the present invention includes a cornealendothelial cell. In addition, even in other cells, for example, in acorneal epithelial cell, a small fraction of basal cells proliferate,and a majority of cells do not proliferate. Therefore, it is possible tostate that even a retinal cell, a vitreous body cell, a cornealepithelial cell, and a corneal parenchymal cell other than a cornealendothelial cell have an extremely important meaning from the viewpointof treatment or prevention of an ophthalmological disease.

In a further preferable embodiment, an eye cell which is a subject ofthe present invention includes a corneal endothelial cell of a primate.In a further more preferable embodiment, an eye cell which is a subjectof the present invention includes a human corneal endothelial cell.

In another embodiment, a cell which is a subject of the presentinvention (e.g., eye cell) is in a confluent state. No technique inwhich a cell can proliferate in such a confluent state has previouslybeen reported as far as the present inventors know. Therefore, such atechnique has extremely high usefulness in a point that a cell in anunprecedented category can be proliferated, from the viewpoint oftreatment or prevention of a disease or a disorder.

In a preferable embodiment, the present invention can utilize a cornealendothelial cell in the confluent state. In the confluent state, inordinary culturing, it is observed that the form of a cell becomesfibroblast-like. It is one important characteristic of the presentinvention that even a corneal endothelial cell in the confluent statecan further proliferate and differentiation can be further suppressed,and this means that a cell having a high corneal endothelium densitywhich is an important prognosis determination factor aftertransplantation can be transplanted, toward clinical application ofcultured corneal endothelium transplantation. A preferable concentrationin that case is not limited, but when R-spondin 1 is used, it can beabout 1 ng/ml to about 100 ng/ml, preferably about 10 ng/ml to about 100ng/ml, further preferably about 10 ng/ml. No technique in which a cellcan proliferate in such a confluent state has previously been reportedas far as the present inventors know. Therefore, such a technique hasextremely high usefulness in a point that a cell in an unprecedentedcategory can be proliferated, from the viewpoint of treatment orprevention of a disease or a disorder. For example, the cell density canbe preferably about 570 cells/mm² or more, about 700 cells/mm² or more,about 800 cells/mm² or more, or about 1000 cells/mm² or more.

As used herein, a “culture” refers to what is produced by culturing acell such as a corneal endothelial cell. Therefore, a “cornealendothelium culture” refers to a culture of the corneal endothelium, andusually refers to a culture which exists in a state different from thatof a cell existing in a living body. As a corneal endothelium cultureobtained by a previous culturing method, at best, only a confluentculture of about 500 cells/mm² was obtained, as shown in Examples.Therefore, from the viewpoint of a culture, it is possible to state thatsuch a cell density was not attained concerning a culture obtained byculturing or subculturing for a long term. That is, a cornealendothelial cell is easily reduced in the density by culturing. Thecorneal endothelium density is clinically one of the most importantindices of degree of health. For this reason, it is important to culturea cell to the high density from the viewpoint of regeneration therapy.In addition, after the endothelium density is increased in advance, thecell can be administered to a living body, and this can be an extremelyimportant therapeutic agent. In this sense, it is important that thereduced density was increased again by an ordinary culturing method. Anormal value of the human corneal endothelium in a living body is around2500 to 3000 cells/mm², and the present invention is meaningful in apoint that it provides a technique of bringing the cell density of aculture close to the range, or making the density exceed the range.

In another embodiment, a cell which is a subject of the presentinvention (e.g., eye cell) can be a corneal tissue itself. Such acorneal tissue itself can be a sclerocornea slice.

Therefore, in another aspect, the present invention provides a cornealendothelium culture, wherein the corneal endothelium exists at a higherdensity than the cell density in the confluent state.

Preferably, the Ki67-positive cell exits at a percentage of about 4% ormore, more preferably at a percentage of about 7% or more, furtherpreferably at a percentage of about 10% or more. Since the ordinaryexistence percentage is around 1%, a corneal tissue containing such aKi67-positive cell, that is, a proliferating cell was not presentpreviously, and the corneal tissue is found to be also valuable as anovel graft for treatment.

The density of the corneal endothelial cell is preferably about 4000cells/mm² or more, more preferably about 4500 cells/mm² or more, furtherpreferably about 5000 cells/mm² or more. An ordinary value of the humancorneal endothelium in a living body is around 2500 to 3000 cells/mm²,and it is understood that a novel tissue in which the ratio of aproliferating cell or an undifferentiated cell, or a corneal endothelialcell is increased to an unprecedented extent is provided by applying anagent for suppressing differentiation and/or promoting proliferation ofthe present invention to a tissue directly. It is understood that such atissue is utilized for improving, treating or preventing a disease, adisorder or a condition of the cornea.

(Preservation or Culturing of Cell)

In another aspect, the present invention provides a composition forpreserving a cell or culturing a cell, comprising the agent forsuppressing differentiation and/or promoting proliferation of thepresent invention. The agent for suppressing differentiation and/orpromoting proliferation used in the present invention may be any agentfor suppressing differentiation and/or promoting proliferation describedin the item of (Differentiation suppression and/or proliferationpromotion) and other items. In addition, a cell which is a subject ofthe present invention is intended for preserving a cell or culturing acell, and it is understood that the cell may be any cell embodimentdescribed in the item of (Differentiation suppression and/orproliferation promotion) and other items. That is, a cell which is asubject of the present invention is not particularly limited, but isintended for preserving a cell or culturing a cell, and includes an eyecell, a nerve cell including a cell derived from a neural crest cell(including a corneal endothelial cell), and an epithelial cell such as aconjunctival epithelial cell, an amniotic epithelial cell, an oralmucosa epithelial cell, a nose mucosa epithelial cell, and a cornealepithelial cell. In a preferable embodiment, a cell which is a subjectof the present invention is an eye cell. An eye cell which is a subjectof the present invention is intended for preserving a cell or culturinga cell, and includes, but is not limited to a retinal cell, a vitreousbody cell, a corneal epithelial cell, a corneal parenchymal cell and acorneal endothelial cell.

In one embodiment of a composition for preserving a cell or culturing acell of the present invention, an eye cell which is a subject of thepresent invention is a cell which does not proliferate in the stationarystate. Not wishing to be bound by any theory, the present inventionexerts an unprecedented effect from the viewpoint of cell preservationor cell culturing in a point that even a cell which does not proliferatein the stationary state can be proliferated by the present invention.

In another embodiment of the composition for preserving a cell orculturing a cell of the present invention, an eye cell which is asubject of the present invention includes at least one kind of cellselected from a retinal cell, a vitreous body cell, a corneal epithelialcell, a corneal parenchymal cell and a corneal endothelial cell.Particularly, a corneal endothelial cell is a cell which proliferateslittle in the stationary state, and a point that this cell can beproliferated has an extremely important meaning from the viewpoint ofcell preservation or cell culturing, and as a result, from the viewpointof treatment or prevention of an ophthalmological disease using a cellto be used. Therefore, in one preferable embodiment, an eye cell whichis a subject of the present invention includes a corneal endothelialcell. In addition, even in other cells, for example, in a cornealepithelial cell, a small fraction of basal cells proliferate, and amajority of cells do not proliferate. Therefore, it is possible to statethat even a retinal cell, a vitreous body cell, a corneal epithelialcell, and a corneal parenchymal cell other than a corneal endothelialcell have an extremely important meaning from the viewpoint of cellpreservation or cell culturing, and as a result, from the viewpoint oftreatment or prevention of an ophthalmological disease by cell treatmentor the like to be used.

In a further preferable embodiment of the composition for preserving acell or culturing a cell of the present invention, an eye cell which isa subject of the present invention includes a corneal endothelial cellof a primate. In a further more preferable embodiment, an eye cell whichis a subject of the present invention includes a human cornealendothelial cell.

In another embodiment of the composition for preserving a cell orculturing a cell of the present invention, a cell which is a subject ofthe present invention (e.g., eye cell) is in the confluent state. Atechnique in which a cell can proliferate in such a confluent state isextremely meaningful also from the viewpoint of cell preservation orcell culturing. Therefore, such a technique has extremely highusefulness in a point that a cell in an unprecedented category can beproliferated, and also from the viewpoint of treatment or prevention ofa disease or a disorder using a cell which is obtained as a result ofcell preservation or cell culturing.

Therefore, in a preferable embodiment, the present invention provides acomposition for preserving the cornea or culturing a corneal endothelialcell, comprising the agent for suppressing differentiation and/orpromoting proliferation of the present invention. Such a composition mayutilize at least one kind of R-spondins, SHH, an agonist of SHH (e.g.,agonist of Frizzled family such as purmorphamine), an agent suppressingGPR49/LGR5 or a functional equivalent thereof which are an activeingredient of the agent for suppressing differentiation and/or promotingproliferation of the present invention, as it is, or can be prepared byaddition of other ingredients. Alternatively, the present inventionprovides a method for preserving or culturing a cell, using the agentfor suppressing differentiation and/or promoting proliferation of thepresent invention. In one embodiment, the present invention provides amethod for preserving the cornea or culturing a corneal endothelialcell, comprising the step of using the agent for suppressingdifferentiation and/or promoting proliferation of the present invention.It should be understood that the agent for suppressing differentiationand/or promoting proliferation contained in the composition forpreserving the cornea or culturing a corneal endothelial cell of thepresent invention can adopt any embodiment described in (Differentiationsuppression and/or proliferation promotion).

In addition, whether a corneal endothelial cell is normally cultured ornot can be determined herein by confirming if a corneal endothelial cellmaintains at least one characteristic such as its inherent function (asused herein, also referred to as “normal function”). Examples of such afunction include ZO-1 and Na⁺/K⁺-ATPase, adaptive capacity to cornealtransplantation (Matsubara M, Tanishima T: Wound-healing of the cornealendothelium in the monkey: a morphometric study, Jpn J Ophthalmol 1982,26: 264-273; Matsubara M, Tanishima T: Wound-healing of cornealendothelium in monkey: an autoradiographic study, Jpn J Ophthalmol 1983,27: 444-450; Van Horn D L, Hyndiuk R A: Endothelial wound repair inprimate cornea, Exp Eye Res 1975, 21: 113-124 and VanHorn D L, Sendele DD, Seideman S, Buco P J: Regenerative capacity of the cornealendothelium in rabbit and cat, Invest Ophthalmol Vis Sci 1977, 16:597-613), but are not limited thereto. That is, it is understood thatthe “normal function” may be an index showing a function necessary forrealizing corneal transplantation, or sufficiency for realizing cornealtransplantation. For example, a method for determining normalization canbe performed by observing a change in expression thereof using afunctional protein in a corneal endothelial cell such as ZO-1 andNa⁺/K⁺-ATPase as an index, or investigating whether the cell isengrafted and functions or not, by transplantation into a monkey or thelike. A determination method by transplantation can be performed asfollows. That is, the corneal endothelium is cultured on type I collagento make a cultured corneal endothelium sheet. Under general anesthesia,limbus corneae of a cynomolgus monkey is incised 1.5 mm, an operationtool made of silicon is inserted into an anterior chamber, and a cornealendothelial cell is mechanically curetted to make a bullous keratopathymodel. Subsequently, limbus corneae is incised 5 to 6 mm, the culturedcorneal endothelium sheet is inserted into the anterior chamber, and theanterior chamber is replaced with the air, thereby, the sheet is adheredto the corneal endothelium surface. The effect of treating bullouskeratopathy by transplantation of the cultured corneal endothelium sheetcan be obtained by evaluating cornea transparency with a slit lampmicroscope.

(Treatment or Prevention of Cell Disorder)

In another aspect, the present invention provides a pharmaceuticalcomposition for treating a disorder of a cell or preventing progressionof the disorder of the cell, comprising the agent for suppressingdifferentiation and/or promoting proliferation of the present invention.The agent for suppressing differentiation and/or promoting proliferationused in the present invention may be any agent for suppressingdifferentiation and/or promoting proliferation described in the item of(Differentiation suppression and/or proliferation promotion) and otheritems. In addition, it is understood that a cell which is a subject ofthe present invention may be any cell embodiment described in the itemof (Differentiation suppression and/or proliferation promotion) andother items, as far as it is intended for treating a disorder of a cellor preventing progression of the disorder of the cell. That is, a cellwhich is a subject of the present invention is not particularly limited,and includes an eye cell, a nerve cell including a cell derived from aneural crest cell (including a corneal endothelial cell), and anepithelial cell such as a conjunctival epithelial cell, an amnioticepithelial cell, an oral mucosa epithelial cell, a nose mucosaepithelial cell, and a corneal epithelial cell, as far as the cell isintended for treating a disorder of a cell or preventing progression ofthe disorder of the cell. In a preferable embodiment, a cell which is asubject of the present invention is an eye cell. An eye cell which is asubject of the present invention includes a retinal cell, a vitreousbody cell, a corneal epithelial cell, a corneal parenchymal cell and acorneal endothelial cell, but is not limited to them.

In one embodiment of the pharmaceutical composition of the presentinvention, an eye cell which is a subject of the present invention is acell which does not proliferate in the stationary state. Not wishing tobe bound by any theory, in a point that even a cell which does notproliferate in the stationary state can be proliferated by the presentinvention, the present invention exerts a meaningful effect in a pointthat treatment of a disorder of a cell or prevention of progression ofthe disorder of the cell can be performed.

In another embodiment of the pharmaceutical composition of the presentinvention, an eye cell which is a subject of the present inventionincludes at least one kind of cell selected from a retinal cell, avitreous body cell, a corneal epithelial cell, a corneal parenchymalcell and a corneal endothelial cell. Particularly, a corneal endothelialcell is a cell which proliferates little in the stationary state, and apoint that this cell can be proliferated has an extremely importantmeaning from the viewpoint of treatment of a disorder of a cell orprevention of progression of the disorder of the cell, and from theviewpoint of treatment or prevention of an ophthalmologial disease.Therefore, in one preferable embodiment, an eye cell which is a subjectof the present invention includes a corneal endothelial cell. Inaddition, even in other cells, for example, in a corneal epithelialcell, a small fraction of basal cells proliferate, and a majority ofcells do not proliferate. Therefore, it is possible to state that even aretinal cell, a vitreous body cell, a corneal epithelial cell, and acorneal parenchymal cell other than a corneal endothelial cell have anextremely important meaning from the viewpoint of treatment orprevention of an ophthalmological disease, in light of a viewpoint oftreatment of a disorder of a cell or prevention of progression of thedisorder of the cell.

In a further preferable embodiment of the pharmaceutical composition ofthe present invention, an eye cell which is a subject of the presentinvention includes a corneal endothelial cell of a primate. In a furthermore preferable embodiment, an eye cell which is a subject of thepresent invention includes a human corneal endothelial cell.

In another embodiment of the pharmaceutical composition of the presentinvention, a cell which is a subject of the present invention (e.g., eyecell) is in the confluent state. A technique in which a cell canproliferate in such a confluent state is meaningful also from theviewpoint of treatment of a disorder of a cell or prevention ofprogression of the disorder of the cell. Therefore, such a technique hasextremely high usefulness in a point that a cell of an unprecedentedcategory can be proliferated, from the viewpoint of treatment of adisorder of a cell or prevention of progression of the disorder of thecell.

In a preferable embodiment, the present invention provides apharmaceutical composition for treating a corneal endothelial celldisorder or preventing progression of a corneal endothelial celldisorder, comprising the agent for suppressing differentiation and/orpromoting proliferation of the present invention. It should beunderstood that the agent for suppressing differentiation and/orpromoting proliferation contained in the pharmaceutical composition ofthe present invention can adopt any embodiment described in(Differentiation suppression and/or proliferation promotion).

As used herein, “treatment”, concerning a certain disease or disorder(e.g., corneal disease), refers to that, when such a condition occurs,worsening of such a disease or disorder is prevented, preferably thecurrent condition is maintained, more preferably the disease or thedisorder is alleviated, further preferably it is eliminated.

As used herein, “prevention”, concerning a certain disease or disorder,refers to avoid such a condition before it occurs. The agent of thepresent invention can be used to perform diagnosis, and if necessary,the agent of the present invention can be used to, for example, preventa corneal disease or the like, or take a precaution.

When the effect of the therapeutic agent of the present invention isconfirmed, determination can be conducted by observing adaptive capacityto corneal transplantation. Concerning adaptive capacity to cornealtransplantation, generally a transplantation test of a cultured cell canbe performed by mechanically curetting the corneal endothelium, as abullous keratopathy model, in an experimental animal such as a rabbit.However, since a corneal endothelial cell of a rabbit proliferates in aliving body, a possibility of spontaneous recovery due to proliferationof a corneal endothelial cell of a host cannot be denied (Matsubara M,et al., Jpn J Ophthalmol 1982, 26: 264-273; Matsubara M, et al., Jpn JOphthalmol 1983, 27: 444-450; Van Horn D L, et al., Exp Eye Res 1975,21: 113-124 and Van Horn D L, et al., Invest Ophthalmol Vis Sci 1977,16: 597-613). Therefore, in order to evaluate the adaptive capacity totransplantation more accurately, it is preferable to evaluateengraftment in a primate. When adaptive capacity to transplantation inhuman is evaluated, adaptability is evaluated after at least 1 month,preferably at least 2 months, more preferably at least 3 months, furtherpreferably at least 6 months, further more preferably at least 12 monthsin a cynomolgus monkey or the like which is a primate. It isparticularly important in application to human to confirm the adaptivecapacity to transplantation in a primate such as a monkey.

When the present invention is administered as a pharmaceutical, avariety of delivery systems are known, and the therapeutic agent of thepresent invention can be administered using such a system. Examples ofsuch a system include encapsulation into a liposome, a microparticle,and a microcapsule; use of a recombinant cell which can express atherapeutic agent (e.g., polypeptide), use of endocytosis mediated witha receptor; and construction of a therapeutic nucleic acid as a part ofa retrovirus vector or another vector. Examples of the administrationmethod include, but are not limited to, intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, extradural andoral routes. It is also possible to administer a pharmaceutical by anyof preferable routes, for example, by infusion, by bolus injection, byinhalation through an epithelium or skin mucosal lining (e.g., oralcavity, rectum, and intestinal mucosa), and, if necessary, an inhaler ora sprayer can be employed using an aerosol agent, and the pharmaceuticalcan also be administered together with other biologically active agents.Administration can be systemic or local. Further, when the presentinvention is used in the ophthalmological field, the pharmaceutical canbe administered by any of appropriate routes, such as direct infusioninto an eye.

In the case of treatment of the cornea or the like, a conventionaltechnique such as ocular instillation, eye ointment application,subconjunctival injection, and vitreous body injection can be used.

In a specific aspect in which a therapeutic agent is a nucleic acidencoding a protein, expression of an encoded protein can be promoted byconstructing the nucleic acid as a part of an appropriate nucleic acidexpression vector, and administering the nucleic acid so as to exist ina cell for in vivo application. This can be performed, for example, byuse of a retrovirus vector, by direct injection, by use of amicroparticle gun, by coating a nucleic acid with a lipid, a cellsurface receptor or a transfection agent, or by administering a nucleicacid connected to a tag sequence which is known to enter a nucleus.Alternatively, the nucleic acid therapeutic agent can be introduced intoa cell, and, for expression, can be taken into a host cell DNA byhomologous recombination.

Generally, the pharmaceutical, the therapeutic agent, the preventiveagent or the like of the present invention comprises a therapeuticallyeffective amount of a therapeutic agent or active ingredient, and apharmaceutically acceptable carrier. As used herein, “pharmaceuticallyacceptable” means that the pharmaceutical, agent or the like is approvedby a governmental regulatory authority, or listed in pharmacopoeia orother generally accepted pharmacopoeias, for use in an animal, moreparticularly, human. A “carrier” as used herein refers to a diluent, anadjuvant, an excipient, or a vehicle which is administered together witha therapeutic agent. Such a carrier can be a sterile liquid, forexample, water and an oil, includes carries derived from a petroleum, ananimal, a plant or a synthetic origin, and includes a peanut oil, asoybean oil, a mineral oil, and a sesame oil without limitation. Whenthe pharmaceutical is orally administered, water is a preferablecarrier. When the pharmaceutical composition is administeredintravenously, physiological saline and aqueous dextrose are preferablecarriers. Preferably, a physiological saline solution, as well asaqueous dextrose and a glycerol solution are used for a liquid carrierof an injectable solution. An appropriate excipient includes lightsilicic anhydride, crystalline cellulose, mannitol, starch, glucose,lactose, sucrose, gelatin, malt, rice, wheat flower, chalk, silica gel,sodium stearate, glyceryl monostearate, talc, sodium chloride, powderedskim milk, glycerol, propylene, glycol, water, ethanol, carmellosecalcium, carmellose sodium, hydroxypropylcellulose,hydroxypropylmethylcellulose, polyvinyl acetal diethyl aminoacetate,polyvinylpyrrolidone, gelatin, medium chain fatty acid tryglyceride,polyoxyethylene hydrogenated castor oil 60, white sugar,carboxymethylcellulose, corn starch, and an inorganic salt. Thecomposition, when desirable, can contain a small amount of a wettingagent or emulsifying agent, or a pH buffer. These compositions can takethe form of a solution, a suspension, an emulsion, a tablet, a pill, acapsule, a powder, a sustained-release formulation or the like. Using atraditional binder and a carrier, for example, triglyceride, thecomposition can also be formulated as a suppository. An oral formulationcan also contain a standard carrier such as pharmaceutical grademannitol, lactose, starch, magnesium stearate, saccharin sodium,cellulose, and magnesium carbonate. Examples of an appropriate carrierare described in E. W. Martin, Remington's Pharmaceutical Sciences (MarkPublishing Company, Easton, U.S.A.). Such a composition contains atherapeutically effective amount of a therapeutic agent, preferably, apurified type of a therapeutic agent together with an appropriate amountof a carrier so as to provide an adequate dosage form to a patient. Aformulation must be suitable for an administration mode. In addition,for example, a surfactant, an excipient, a coloring agent, a flavoringagent, a preservative, a stabilizer, a buffer, a suspending agent, atonicity agent, a binder, a disintegrating agent, a lubricant, a flowpromoter, and a taste masking agent may be contained.

In a preferable embodiment, a composition can be formulated as apharmaceutical composition adapted to injection administration to humanaccording to a known method. Representatively, a composition forinjection administration is a solution in a sterile isotonic aqueousbuffer. If necessary, the composition can also contain a solubilizer anda local anesthetic such as lidocaine which mitigates a pain at aninjection site. Generally, ingredients can be supplied as a lyophilizedpowder or a water-free concentrate by supplying the ingredientsseparately, or by mixing them together in a unit dosage form in a sealedcontainer such as an ampoule or a sachet indicating the amount of anactive agent. When the composition is to be administered by infusion,the composition can also be dispensed using an infusion bottlecontaining a sterile pharmaceutical grade water or physiological saline.When the composition is to be administered by injection, an ampoule ofsterile water or physiological saline for injection can be provided sothat the ingredients can be mixed prior to administration.

The pharmaceutical, the therapeutic agent, or the preventive agent ofthe present invention can also be formulated with another prodrug (e.g.,ester) of a neutral type or a salt type. A pharmaceutically acceptablesalt includes salts formed with a free carboxyl group derived fromhydrochloric acid, phosphoric acid, acetic acid, oxalic acid andtartaric acid, salts formed with a free amine group derived fromisopropylamine, triethylamine, 2-ethylaminoethanol, histidine, andprocaine, as well as salts derived from sodium, potassium, ammonium,calcium, and ferric hydroxide.

The amount of the therapeutic agent of the present invention effectivefor treating a specific disorder or condition can vary depending on thenature of the disorder or the condition, but a person skilled in the artcan determine the amount by a standard clinical technique based on thedescriptions described herein. Further, depending on the situation, itis also possible to assist identification of an optimal dose range usingan in vitro assay. Since the precise dose to be used in a formulationcan also vary depending on an administration route, and severity of adisease or a disorder, the dose should be determined according to thejudgment of an attending physician and the situation of each patient.However, a dose range suitable for direct administration to the corneais generally about 1 to 500 micrograms of an active ingredient perkilogram weight, but is not limited to this, and can be smaller orlarger than this range. An effective dose can be presumed from adose-response curve obtained from an in vitro or animal model testsystem.

The pharmaceutical composition, the therapeutic agent or the preventiveagent of the present invention can be provided as a kit.

As used herein, a “kit” refers to a unit which is usually divided intotwo or more sections, and which provides a part to be provided (e.g.,therapeutic agent, antibody, label, and direction). For the purpose ofproviding a composition which should not be provided in the form of amixture but is preferably used by mixing immediately before use, thisform of kit is preferable. It is advantageous that such a kit ispreferably provided with a part to be provided (e.g., instruction ordirection describing how to use the therapeutic agent, or how to treatthe reagent). As used herein, when the kit is used as a reagent kit, thekit usually includes an instruction describing how to use an antibody.

As used herein, an “instruction” has a description explaining how to usethe present invention to a doctor or other users. This instructiondescribes wording instructing the reader on the detection method of thepresent invention, how to use a diagnostic, or administration of apharmaceutical or the like. In addition, the instruction may describewording instructing the reader on administration to a skeletal muscle(e.g., by injection) as an administration site. This instruction iswritten according to the format defined by a regulatory authority of acountry where the present invention is implemented (e.g., Ministry ofHealth, Labour and Welfare in Japan and Food and Drug Administration(FDA) in the U.S.), and explicitly describes that the pharmaceutical wasapproved by the regulatory authority. The instruction is the so-calledpackage insert, and is usually provided in a paper medium, but is notlimited thereto. For example, the instruction can be provided in theform such as an electronic medium (e.g., homepage provided on theinternet and electronic mail).

In a specific embodiment, the present invention provides apharmaceutical pack or kit, comprising one or more containers filledwith one or more ingredients of the pharmaceutical composition of thepresent invention. Depending on the situation, it is possible toindicate on such a container information showing approval by agovernmental organization which regulates manufacture, use or sales of apharmaceutical or a biological product in relation to manufacture, useor sales for administration to human, in a form defined by thegovernmental organization.

The kit of the present invention can also contain an expression vectorencoding a protein used as the therapeutic agent, the preventive agentor the agent of the present invention, and this protein can also bereconstituted in order to form a biologically active complex afterexpression. Such a kit preferably also contains a necessary buffer and anecessary reagent. Depending on the situation, it is possible toindicate on such a container a direction for the use of the kit and/orinformation showing approval by a governmental organization whichregulates manufacture, use or sales of a pharmaceutical or a biologicalproduct in relation to manufacture, use or sales for administration tohuman, in a form defined by the governmental organization.

In a specific embodiment, the pharmaceutical composition comprising anucleic acid of the present invention can be administered by means of aliposome, a microparticle, or a microcapsule. In various aspects of thepresent invention, it may be useful to attain sustained release of anucleic acid using such a composition.

In another aspect, treatment of the present invention can be implementedusing a corneal tissue itself prepared using the present invention, forexample, a sclerocornea slice. Preferably, the Ki67-positive cell exitsat a ratio of about 4% or more, more preferably at a ratio of about 7%or more, further preferably at a ratio of about 10% or more. Since theordinary existence ratio is around 1%, a corneal tissue containing sucha Ki67-positive cell, that is, a proliferating cell was not presentpreviously, and a therapeutic effect better than that of a cornealtissue which is directly transplanted from a living body, as a graft fornovel treatment, is expected.

The density of the corneal endothelial cell is preferably about 4000cells/mm² or more, more preferably about 4500 cells/mm² or more, furtherpreferably about 5000 cells/mm² or more. A normal value of the humancorneal endothelium in a living body is around 2500 to 3000 cells/mm².It is understood that a novel tissue in which the ratio of aproliferating cell or an undifferentiated cell, or a corneal endothelialcell is increased to an unprecedented extent is provided by directlyapplying the agent for suppressing differentiation and/or promotingproliferation of the present invention to a tissue. It is understoodthat such a tissue is utilized for improving, treating or preventing adisease, a disorder or a condition of the cornea for the purpose ofexerting a therapeutic effect better than that of a corneal tissue whichis directly transplanted from a living body.

(Cell Treatment)

In another aspect, the present invention provides a therapeutic agent ora progression preventive agent for a disorder of a cell, or a disease ora disorder due to the cell disorder, comprising a cell which is culturedusing the agent for suppressing differentiation and/or promotingproliferation of the present invention. The agent for suppressingdifferentiation and/or promoting proliferation used in the presentinvention may be any agent for suppressing differentiation and/orpromoting proliferation described in the item of (Differentiationsuppression and/or proliferation promotion) and other items. Inaddition, it is understood that a cell which is a subject of the presentinvention may be any cell embodiment described in the item of(Differentiation suppression and/or proliferation promotion) and otheritems. That is, examples of the cell which is a subject of the presentinvention may include, but are not particularly limited to, an eye cell,a nerve cell including a cell derived from a neural crest cell(including a corneal endothelial cell), and epithelial cells such as aconjunctival epithelial cell, an amniotic epithelial cell, an oralmucosa epithelial cell, a nose mucosa epithelial cell, and a cornealepithelial cell. In a preferable embodiment, a cell which is a subjectof the present invention is an eye cell. An eye cell which is a subjectof the present invention can include a retinal cell, a vitreous bodycell, a corneal epithelial cell, a corneal parenchymal cell and acorneal endothelial cell, but is not limited to them.

In one embodiment of the therapeutic agent or the progression preventiveagent of the present invention, an eye cell which is a subject of thepresent invention is a cell which does not proliferate in the stationarystate. Not wishing to be bound by any theory, the present inventionexerts an unprecedented effect in a point that even a cell which doesnot proliferate in the stationary state can be proliferated by thepresent invention.

In another embodiment of the therapeutic agent or the progressionpreventive agent of the present invention, an eye cell which is asubject of the present invention includes at least one kind of cellselected from a retinal cell, a vitreous body cell, a corneal epithelialcell, a corneal parenchymal cell and a corneal endothelial cell.Particularly, a corneal endothelial cell is a cell which proliferateslittle in the stationary state, and a point that this cell can beproliferated has an extremely important meaning from the viewpoint oftreatment or prevention of an ophthalmological disease. Therefore, inone preferable embodiment, an eye cell which is a subject of the presentinvention includes a corneal endothelial cell. In addition, even inother cells, for example, in a corneal epithelial cell, a small fractionof basal cells proliferate, and a majority of cells do not proliferate.Therefore, it is possible to state that even a retinal cell, a vitreousbody cell, a corneal epithelial cell and a corneal parenchymal cellother than a corneal endothelial cell have an extremely importantmeaning from the viewpoint of treatment or prevention of anophthalmological disease.

In a further preferable embodiment of the therapeutic agent or theprogression preventive agent of the present invention, an eye cell whichis a subject of the present invention includes a corneal endothelialcell of a primate. In a further more preferable embodiment, an eye cellwhich is a subject of the present invention includes a human cornealendothelial cell.

In another embodiment of the therapeutic agent or the progressionpreventive agent of the present invention, a cell which is a subject ofthe present invention (e.g., eye cell) is in the confluent state. Notechnique in which a cell can proliferate in such a confluent state haspreviously been reported as far as the present inventors know.Therefore, such a technique has extremely high usefulness in a pointthat a cell in an unprecedented category can be proliferated, from theviewpoint treatment or prevention of a disease or a disorder.

In one embodiment, the present invention provides a therapeutic agent ora progression preventive agent for a corneal endothelial cell disorder,comprising a corneal endothelial cell which was cultured using the agentfor suppressing differentiation and/or promoting proliferation of thepresent invention. It should be understood that the agent forsuppressing differentiation and/or promoting proliferation contained inthe therapeutic agent or the progression preventive agent of the presentinvention can adopt any embodiment described in (Differentiationsuppression and/or proliferation promotion), (Treatment or prevention ofcell disorder), (Preservation or culturing of cell) and the like.

In one embodiment, particularly, in the case of a corneal endothelialcell, a cell contained in the therapeutic agent or the progressionpreventive agent of the present invention exists as a populationincluding more cells which have a higher cell density than that ofnormal cells which exist in a natural state and/or are undifferentiated.Since a therapeutic agent or a progression preventive agent containingsuch a cell was unable to be produced before, the present invention hashigh clinical usefulness in that a disease or a disorder which wasunable to be treated or prevented, or difficult to treat or preventbefore (e.g., including corneal endothelial diseases such as bullouskeratopathy and Fuchs endothelial corneal dystrophy without beinglimited to them) can be treated.

The therapeutic agent or the preventive agent of the present inventioncan be produced by culturing a cell such as a corneal endothelial cellusing the agent for suppressing differentiation and/or promotingproliferation of the present invention. In such a case, theproliferation ability or the differentiation level can be determinedusing, as an index, GPR49/LGR5, and/or the factors of the Hedgehogpathway such as SHH, PTCH1, GLI1, and GLI2, the factors of the Wntpathway such as LRP6 and β-catenin, or known differentiation markerssuch as Ki-67 and BrdU described in another aspect of the presentinvention. Determination of the proliferation ability or thedifferentiation level is explained in detail separately describedherein.

The present invention provides a process for producing a therapeuticagent or a progression preventive agent comprising the following steps:(A) a step of providing a cell such as a corneal endothelial cell; (B) astep of contacting the agent for suppressing differentiation and/orpromoting proliferation of the present invention with the cell; and (C)a step of culturing the cell which was brought into contact in the step(B). Further, if necessary, the process may include a step ofdetermining the proliferation ability or the differentiation levelusing, as an index, GPR49/LGR5, and/or the factors of the Hedgehogpathway such as SHH, PTCH1, GLI1, and GLI2, the factors of the Wntpathway such as LRP6 and β-catenin, or known differentiation markerssuch as Ki-67 and BrdU described in another aspect of the presentinvention, and selecting a cell having a high differentiation abilityand a high proliferation ability.

According to the present invention, a method for treating or preventinga corneal endothelial disease, disorder, or condition, comprising a stepof administering the agent for suppressing differentiation and/orpromoting proliferation of the present invention to a subject in need oftreatment, or a step of administering the therapeutic agent or theprogression preventive agent of the present invention to a subject inneed of treatment. Since the step of administering the therapeutic agentor the progression preventive agent of the present invention to asubject in need of treatment is an act of administering a pharmaceuticalcontaining a cell, it may be called transplantation.

According to the method of treatment or the method of prevention of thepresent invention, a corneal endothelial disease, disorder or conditioncan be prevented and/or treated, since an administered or transplantedcell cures or restores damage of the corneal endothelium.

In implementation of the method of treatment in accordance with thepresent invention, a cell which can contain a corneal endothelial cellcan be obtained from a mammal including human, preferably, an individualitself undergoing transplantation or an aborted fetus.

(Marker for Identifying Differentiation Ability and Proliferation ofGPR49/LGR5, and/or Factors of Hedgehog Pathway Such as SHH, PTCH1, GLI1,and GLI2, and Factors of Wnt Pathway Such as LRP6 and β-Catenin)

In one aspect, the present invention provides a marker for identifying acell having a high proliferation ability among corneal endothelial cellsand/or the differentiation ability of a corneal endothelial cell,comprising GPR49/LGR5, and/or the factors of the Hedgehog pathway suchas SHH, PTCH1, GLI1, and GLI2, and/or the factors of the Wnt pathwaysuch as LRP6 and β-catenin. GPR49/LGR5, the factors of the Hedgehogpathway such as SHH, PTCH1, GLI1, and GLI2 and the factors of the Wntpathway such as LRP6 and β-catenin exist in a living body, and it wasfound in the present invention that they can be used as an index markerof a cell having a high proliferation ability and/or the differentiationability.

In one embodiment, a cell having a high proliferation ability which is asubject of the present invention is an undifferentiated cell.

In another embodiment, a cell having a high proliferation ability whichis a subject of the present invention is a stem cell.

In another embodiment, a corneal endothelial cell which is a subject ofthe present invention is a human cell.

In still another embodiment, the proliferation ability of a cornealendothelial cell which is a subject of the present invention isidentified by a characteristic selected from the group consisting ofcolony forming ability, Ki-67 positivity and BrdU positivity.

Expression of GPR49/LGR5, and/or the factors of the Hedgehog route suchas SHH (gene), PTCH1, GLI1, and GLI2 serves as an index of a cell havinga high proliferation ability among corneal endothelial cells and/or thedifferentiation ability of a corneal endothelial cell. Therefore,according to the present invention, by defecting expression ofGPR49/LGR5 and/or SHH, a cell having a high proliferation ability (anundifferentiated cell, a precursor cell or a stem cell) among cornealendothelial cells and/or the differentiation ability of a cornealendothelial cell can be detected or selected. On the other hand,expression of the factors of the Wnt pathway such as LRP6 and β-cateninserves as an index of a cell having a low proliferation ability amongcorneal endothelial cells and/or the differentiation ability of acorneal endothelial cell. Therefore, according to the present invention,by detecting expression of the factors of the Wnt pathway such as LRP6and β-catenin, a cell having a low proliferation ability (a relativelydifferentiated cell, which is not an undifferentiated cell, a precursorcell or a stem cell) among corneal endothelial cells and/or a lowdifferentiation ability of a corneal endothelial cell can be detected orselected. Alternatively, when suppression or disappearance of expressionof the factors of the Wnt pathway such as LRP6 and β-catenin has beendetected, a cell having a high proliferation ability (anundifferentiated cell, a precursor cell or a stem cell) among cornealendothelial cells and/or the differentiation ability of a cornealendothelial cell can be detected or selected.

In another aspect, the present invention provides a detection agent or adiagnostic agent for identifying a cell having a high proliferationability among corneal endothelial cells and/or the differentiationability of a corneal endothelial cell, comprising a substance whichbinds to, or interacts with GPR49/LGR5, and/or the factors of theHedgehog pathway such as Sonic hedgehog (SHH), PTCH1, GLI1, and GLI2,and/or the factors of the Wnt pathway such as LRP6 and β-catenin. Forsuch detection or diagnosis, it is preferable that binding of thesubstance is specific.

As such a detection agent or diagnostic, any substance may be utilizedas far as it can bind to, or interact with GPR49/LGR5, and/or thefactors of the Hedgehog pathway such as Sonic hedgehog (SHH), PTCH1,GLI1, and GLI2, and/or the factors of the Wnt pathway such as LRP6 andβ-catenin Representative examples thereof include an antibody of thesefactors or a fragment or functional equivalent thereof, or a nucleicacid primer or a probe concerning a nucleic acid encoding these factors,but are not limited to them.

The detection agent or the diagnostic agent of the present invention canbe utilized as a detection kit or a diagnosis kit.

In one embodiment, a cell having a high proliferation ability which is asubject of detection or diagnosis of the present invention is anundifferentiated cell.

In another embodiment, a cell having a high proliferation ability whichis a subject of detection or diagnosis of the present invention is astem cell.

In another embodiment, a corneal endothelial cell which is a subject ofdetection or diagnosis of the present invention is a human cell.

In still another embodiment, the proliferation ability of a cornealendothelial cell which is a subject of detection or diagnosis of thepresent invention is identified by a characteristic selected from thegroup consisting of colony forming ability, Ki-67 positivity and BrdUpositivity.

The detection agent of the present invention may be a complex or acomposite molecule in which another substance (e.g., label) is bound toa portion which allows for detection (e.g., antibody). As used herein, a“complex” or a “composite molecule” means any constituent comprising twoor more parts. For example, when one of the parts is a polypeptide, theother part may be a polypeptide, or may be another substance (e.g., asugar, a lipid, a nucleic acid, or a different hydrocarbon). As usedherein, two or more parts constituting the complex may be bound with acovalent bond, or may be bound with another bond (e.g., a hydrogen bond,an ionic bond, hydrophobic interaction, or Van der Waals force). Whentwo or more parts are each a polypeptide, this can also be named as achimeric polypeptide. Therefore, as used herein, a “complex” includes amolecule obtained by connecting a plurality of kinds of molecules suchas a polypeptide, a polynucleotide, a lipid, a sugar, and a lowmolecule.

As used herein, “interaction”, when referring to two substances, refersto that a force (e.g., intermolecular force (Van der Waals force), ahydrogen bond, or hydrophobic interaction) is exerted between onesubstance and the other substance. Usually, the two substances whichhave interacted with each other are in an associated or bonded state.

The term “bond”, as used herein, means physical interaction or chemicalinteraction between two substances, or between combinations thereof. Thebond includes an ionic bond, a non-ionic bond, a hydrogen bond, a Vander Waals bond, and hydrophobic interaction. Physical interaction (bond)can be direct or indirect, and indirect interaction arises through ordue to the effect of another protein or compound. A direct bond does notoccur through or due to the effect of another protein or compound, andrefers to interaction accompanying no other substantial chemicalintermediate.

Therefore, as used herein, an “agent” (or a factor, a detection agent orthe like) which “specifically” interacts with (or binds to) a biologicalagent such as a polynucleotide or a polypeptide includes an agent whoseaffinity for a biological agent such as a polynucleotide or apolypeptide is representatively equal to or higher than, preferablysignificantly (e.g., statistically significantly) higher than theaffinity for other irrelevant (particularly, identity is less than 30%)polynucleotide or polypeptide. Such affinity can be measured, forexample, by a hybridization assay, a binding assay or the like.

As used herein, that a first substance or agent “specifically” interactswith (or binds to) a second substance or agent refers to that a firstsubstance or agent interacts with (or binds to) a second substance oragent with higher affinity than that for a substance or agent other thanthe second substance or agent (particularly, a different substance oragent existing in a sample containing the second substance or agent).Examples of interaction (or bond) specific for a substance or a agentinclude, but are not limited to a ligand-receptor reaction,hybridization in nucleic acids, an antigen-antibody reaction inproteins, and an enzyme-substrate reaction, and when both of a nucleicacid and a protein are involved, a reaction between a transcriptionagent and a binding site of the transcription factor, protein-lipidinteraction, and nucleic acid-lipid interaction. Therefore, when both ofthe substances or agents are nucleic acids, a first substance or agent“specifically interacts” with a second substance or agent including thata first substance or agent has complementarity to at least a part of asecond substance or agent. In addition, for example, when both of thesubstances or agents are proteins, that a first substance or agent“specifically” interacts with (or binds to) a second substance or agentincludes, for example, interaction by an antigen-antibody reaction,interaction by a receptor-ligand reaction, and enzyme-substrateinteraction, but is not limited thereto. When two kinds of substances oragents include a protein and a nucleic acid, that a first substance oragent “specifically” interacts with (or binds to) a second substance oragent includes interaction (or bond) between a transcription factor anda binding region of a nucleic acid molecule which is a subject of thetranscription factor.

As used herein, “detection” or “quantitation” of polynucleotide orpolypeptide expression can be attained, for example, using anappropriate method including mRNA measurement and an immunologicalmeasuring method, that includes binding or interaction with a markerdetection agent. Examples of the molecular biological measuring methodinclude a Northern blotting method, a dot blotting method and a PCRmethod. Examples of the immunological measuring method include, as amethod, an ELISA method using a microtiter plate, an RIA method, afluorescent antibody method, a luminescence immunoassay (LIA), animmunoprecipitation method (IP), a single radical immuno-diffusionmethod (SRID), turbidimetric immunoassay (TIA), a Western blottingmethod, and an immunohistological staining method. In addition, as aquantitation method, an ELISA method or an RIA method can be mentioned.Detection or quantitation can also be performed by a genetic analysismethod using an array (e.g., DNA array or protein array). The DNA arrayis widely reviewed in (Cell Technology, separate volume, “DNA Microarrayand Advanced PCR method”, edited by Shujunsha Co., Ltd.). The proteinarray is described in detail in Nat Genet. 2002 December; 32 Suppl:526-532. Examples of a method for analyzing gene expression include, butare not limited to RT-PCR, a RACE method, a SSCP method, animmunoprecipitation method, a two-hybrid system, and in vitrotranslation, in addition to the aforementioned methods. Such additionalanalysis methods are described, for example, in Genome AnalysisExperimental Method, Nakamura Yusuke Lab. Manual, edited by YusukeNakamura, Yodosha Co., Ltd. (2002), the entirety of descriptions in thedocument are incorporated herein by reference.

As used herein, an “expression amount” refers to an amount of expressionof a polypeptide or mRNA in an objective cell, tissue or the like.Examples of such an expression amount include an expression amount atthe protein level of the present polypeptide evaluated by anyappropriate method including an ELISA method, an RIA method, afluorescent antibody method, a Western blotting method, or animmunological measuring method such as an immunohistological stainingmethod using the antibody of the present invention, and an expressionamount at the mRNA level of a polypeptide used in the present inventionwhich is evaluated by any appropriate method including a molecularbiological measuring method such as a Northern blotting method, a dotblotting method, and a PCR method. “Change in an expression amount”means that an expression amount at the protein level or the mRNA levelof a polypeptide used in the present invention, which is evaluated byany appropriate method including the immunological measuring method andthe molecular biological measuring method, is increased or decreased. Bymeasuring an expression amount of a certain marker, a variety ofdetections or diagnoses based on the marker can be performed.

As used herein, “decrease” or “suppression” or a synonym thereof of theactivity or an expression product (e.g., a protein or a transcriptionproduct (such as RNA)) refers to decrease in an amount, nature or theeffect of a specific activity, transcription product or protein, or anactivity that decreases them.

As used herein, “increase” or “activation” or a synonym thereof of theactivity or an expression product (e.g., a protein or a transcriptionproduct (such as RNA)) refers to an increase in an amount, nature or theeffect of a specific activity, transcription product or protein, or anactivity that increases them.

Therefore, it is understood that an agent which performs differentiationmodulation of an eye cell can be detected or screened using themodulation ability such as decrease, suppression, increase or activationof the marker of the present invention as an index.

As used herein, an “antibody” includes, in a broad sense, a polyclonalantibody, a monoclonal antibody, a multi-specific antibody, a chimericantibody, and an anti-idiotype antibody, as well as a fragment thereof,for example, a Fv fragment, a Fab′ fragment, F(ab′)₂ and Fab fragments,as well as other conjugates or functional equivalents produced byrecombination (e.g., chimeric antibody, humanized antibody,multifunctional antibody, bispecific or oligospecific antibody, singlechain antibody, scFV, diabody, sc(Fv)₂ (single chain (Fv)₂), andscFv-Fc). Furthermore, such an antibody may be covalently bound, orrecombinantly fused with an enzyme, for example, alkaline phosphatase,horseradish peroxidase, or α galactosidase. An anti-GPR49/LGR5 antibodyand a Sonic hedgehog (SHH) antibody used in the present invention may bebound to GPR49/LGR5 and Sonic hedgehog (SHH) proteins, respectively,regardless of the origin, kind, shape or the like thereof. Specifically,known antibodies such as a non-human animal antibody (e.g., a mouseantibody, a rat antibody, or a camel antibody), a human antibody, achimeric antibody, and a humanized antibody can be used. In the presentinvention, a monoclonal or polyclonal antibody can be utilized as anantibody, and preferred is a monoclonal antibody. It is preferable thatbinding of an antibody to each protein of GPR49/LGR5, the factors of theHedgehog pathway such as Sonic hedgehog (SHH), PTCH1, GLI1, and GLI2,and the factors of the Wnt pathway such as LRP6 and β-catenin isspecific binding.

As used herein, an “antigen” refers to any substrate which can bespecifically bound with an antibody molecule. As used herein, an“immunogen” refers to an antigen which can initiate lymphocyteactivation that produces an antigen-specific immunological response. Asused herein, an “epitope” or an “antigen determinant” refers to a sitein an antigen molecule to which an antibody or a lymphocyte receptorbinds. A method for determining an epitope is well-known in the art.When a primary sequence of a nucleic acid or an amino acid is provided,such an epitope can be determined by a person skilled in the art usingthe well-known conventional technique.

As used herein, “means” refers to any matter which can serve as any toolfor attaining a certain object (e.g., detection, diagnosis, ortreatment). Particularly, as used herein, “means which selectivelyrecognizes” refers to means which can recognize a certain subjectdifferently from others.

It is understood that, as an antibody used herein, an antibody of anyspecificity may be used as far as pseudopositivity is decreased.Therefore, an antibody used in the present invention may be a polyclonalantibody, or may be a monoclonal antibody. As used herein, a “ligand”refers to a substance specifically binding to a certain protein.Examples of the ligand include lectin, an antigen, an antibody, ahormone, and a neurotransmitter which specifically bind to a variety ofreceptor protein molecules existing on a cell membrane.

The detection agent or the diagnostic agent or other pharmaceuticals ordrugs of the present invention can take a form of a probe and a primer.The probe and the primer of the present invention can specificallyhybridize with GPR49/LGR5, R-spondins, the factors of the Hedgehogpathway such as Sonic hedgehog (SHH), PTCH1, GLI1, and GLI2, and/or thefactors of the Wnt pathway such as LRP6 and β-catenin. As used herein,expression of GPR49/LGR5, the factors of the Hedgehog pathway such asSonic hedgehog (SHH), PTCH1, GLI1, and GLI2, and/or the factors of theWnt pathway such as LRP6 and β-catenin is an index of the proliferationability in a corneal endothelial cell, and is also useful as an index ofthe differentiated state. Therefore, the probe and the primer inaccordance with the present invention can be used for identifying a cellhaving a high proliferation ability among corneal endothelial cellsand/or the differentiation ability of a corneal endothelial cell. Theprobe and the primer of the present invention, in one embodiment, maydetect expression of GPR49/LGR5, the factors of the Hedgehog pathwaysuch as Sonic hedgehog (SHH), PTCH1, GLI1, and GLI2, and/or the factorsof the Wnt pathway such as LRP6 and β-catenin, and refer to a polymerformed of a plurality of nucleotides or nucleotide pairs such asdeoxyribonucleic acids (DNAs) or ribonucleic acids (RNAs). It is knownthat a double-stranded cDNA can also be utilized in tissue in situhybridization, and the probe and the primer of the present inventionalso include such a double-stranded cDNA. Examples of the probe and theprimer particularly preferable in detection of RNA in a tissue includean RNA probe (riboprobe).

As used herein, a “(nucleic acid) primer” refers to a substancenecessary for initiation of a reaction of a polymer compound to besynthesized, in a polymer synthesizing enzyme reaction. In a reaction ofsynthesizing a nucleic acid molecule, a nucleic acid molecule (e.g., DNAor RNA) complementary to a part of a sequence of a polymer compound tobe synthesized can be used. As used herein, a primer can be used as amarker detection means.

Examples of a nucleic acid molecule which is usually used as a primerinclude molecules having a nucleic acid sequence having a length of atleast 8 consecutive nucleotides, which is complementary to a nucleicacid sequence of an objective gene. Such a nucleic acid sequence can bepreferably a nucleic acid sequence of a length of at least 9 consecutivenucleotides, more preferably a length of at least 10 consecutivenucleotides, further preferably a length of at least 11 consecutivenucleotides, a length of at least 12 consecutive nucleotides, a lengthof at least 13 consecutive nucleotides, a length of at least 14consecutive nucleotides, a length of at least 15 consecutivenucleotides, a length of at least 16 consecutive nucleotides, a lengthof at least 17 consecutive nucleotides, a length of at least 18consecutive nucleotides, a length of at least 19 consecutivenucleotides, a length of at least 20 consecutive nucleotides, a lengthof at least 25 consecutive nucleotides, a length of at least 30consecutive nucleotides, a length of at least 40 consecutivenucleotides, or a length of at least 50 consecutive nucleotides. Anucleic acid sequence used as a probe includes a nucleic acid sequencewhich is at least 70% homologous, more preferably at least 80%homologous, further preferably at least 90% homologous, or at least 95%homologous to the aforementioned sequences. A sequence appropriate as aprimer can vary depending on the nature of a sequence which is intendedto be synthesized (amplified), and a person skilled in the art canappropriately design a primer depending on the intended sequence. Designof such a primer is well-known in the art, and may be performedmanually, or may be performed using a computer program (e.g., LASERGENE,PrimerSelect, or DNAStar).

The primer in accordance with the present invention can also be used asa primer set consisting of two or more of the primers.

The primer and the primer set in accordance with the present inventioncan be utilized as a primer and a primer set according to a conventionalmethod, in a known method for detecting an objective gene utilizing anucleic acid amplification method such as a PCR method, an RT-PCRmethod, a real time PCR method, an in situ PCR method, or a LAMP method.

The primer set in accordance with the present invention can be selectedso that a nucleotide sequence of an objective protein such asGPR49/LGR5, R-spondins, the factors of the Hedgehog pathway such asSonic hedgehog (SHH), PTCH1, GLI1, and GLI2, and/or the factors of theWnt pathway such as LRP6 and β-catenin can be amplified by a nucleicacid amplification method such as a PCR method. The nucleic acidamplification method is well-known, and selection of a primer pair inthe nucleic acid amplification method is obvious to a person skilled inthe art. For example, in a PCR method, primers can be selected so thatone of the two primers (primer pair) pairs with a plus chain ofdouble-stranded DNA of an objective protein such as GPR49/LGR5,R-spondins, the factors of the Hedgehog pathway such as SHH, PTCH1,GLI1, and GLI2, and/or the factors of the Wnt pathway such as LRP6 andβ-catenin, the other primer pairs with a minus chain of thedouble-stranded DNA, and the latter primer pairs with an extended chainwhich is extended by the former primer. In addition, in a LAMP method(WO 00/28082), three regions of F3c, F2c and F1c from the 3′ terminalside, and three regions of B1, B2 and B3 from the 5′ terminal side aredefined, respectively, for a target gene, and these six regions can beused to design four kinds of primers. The primer of the presentinvention can be chemically synthesized based on nucleotide sequencesdisclosed herein. Preparation of the primer is well-known, and can beperformed according to, for example, “Molecular Cloning, A LaboratoryManual 2^(nd) ed.” (Cold Spring Harbor Press (1989)), “Current Protocolsin Molecular Biology” (John Wiley & Sons (1987-1997)).

As used herein, a “probe” refers to a substance being means forretrieval, which is used in a biological experiment such as in vitroand/or in vivo screening. Examples thereof include, but are not limitedto a nucleic acid molecule comprising a specified nucleotide sequence ora peptide comprising a specified amino acid sequence, a specificantibody or a fragment thereof. As used herein, the probe can also beused as a marker detection means.

Examples of a nucleic acid molecule which is usually used as a probeinclude a nucleic acid molecule having a nucleic acid sequence of alength of at least 8 consecutive nucleotides, which is homologous orcomplementary to a nucleic acid sequence of an objective gene. Such anucleic acid sequence can be preferably a nucleic acid sequence of alength of at least 9 consecutive nucleotides, more preferably a lengthof at least 10 consecutive nucleotides, further preferably a length ofat least 11 consecutive nucleotides, a length of at least 12 consecutivenucleotides, a length of at least 13 consecutive nucleotides, a lengthof at least 14 consecutive nucleotides, a length of at least 15consecutive nucleotides, a length of at least 20 consecutivenucleotides, a length of at least 25 consecutive nucleotides, a lengthof at least 30 consecutive nucleotides, a length of at least 40consecutive nucleotides, or a length of at least 50 consecutivenucleotides. A nucleic acid sequence used as a probe includes a nucleicacid sequence which is at least 70% homologous, more preferably at least80% homologous, further preferably at least 90% homologous, or at least95% homologous to the aforementioned sequences.

In one embodiment, the detection agent of the present invention can belabeled. Alternatively, the detection agent of the present invention maybe an agent bound to a tag.

As used herein, a “label” refers to an entity (e.g., substance, energy,or electromagnetic wave) for identifying an objective molecule orsubstance from others. Examples of such a labeling method include an RI(radioisotope) method, a fluorescence method, a biotin method, and achemiluminescence method. When a plurality of markers of the presentinvention, or agents or means for capturing them is labeled by afluorescence method, labeling is performed with fluorescent substanceshaving different fluorescence maximum wavelengths. A difference in thefluorescence maximum wavelength is preferably 10 nm or more. When aligand is labeled, any label can be used as far as it does not influenceon the function, and as a fluorescent substance, Alexa™ Fluor isdesirable. Alexa™ Fluor is a water-soluble fluorescent dye obtained bymodifying coumarin, rhodamine, fluorescein, cyanine or the like, is aseries in response to a wide range of fluorescent wavelengths, and isvery stable, bright, and low in pH sensitivity as compared with otherfluorescent dyes of the corresponding wavelengths. Examples of acombination of fluorescent dyes having a fluorescent maximum wavelengthof 10 nm or longer include a combination of Alexa™ 555 and Alexa™ 633and a combination of Alexa™ 488 and Alexa™ 555. When a nucleic acid islabeled, any label can be used as far as it can bind to a base portionthereof, and it is preferable that a cyanine dye (e.g., Cy3 and Cy5 ofCyDye™ series), a rhodamine 6G reagent, 2-acetylaminofluorene (AAF),AAIF (iodine derivative of AAF) or the like is used. Examples of thefluorescent substances having a difference in the fluorescence maximumwavelength of 10 nm or more include a combination of Cy5 and a rhodamine6G reagent, a combination of Cy3 and fluorescein, and a combination of arhodamine 6G reagent and fluorescein. In the present invention, such alabel can be utilized to alter an objective subject so that it can bedetected with a detection means to be used. Such an alteration is knownin the art, and a person skilled in the art can appropriately carry outsuch a method in accordance with a label and an objective subject.

As used herein, a “tag” refers to a substance for selecting a moleculeby a specific recognizing mechanism such as receptor-ligand, morespecifically, a substance which plays a role of a binding partner forbinding to a specified substance (e.g., a substance having arelationship such as biotin-avidin or biotin-streptavidin), and can beincluded in the category of “label”. Hence, for example, a specifiedsubstance with a tag bound thereto can be selected by contacting thesubstance with a substrate with a binding partner of a tag sequencebound thereto. Such a tag or label is well-known in the art.Representative tag sequences include a myc tag, a His tag, HA, and anAvi, but are not limited to them. The marker or the marker detectionagent of the present invention may be bound to such a tag.

In one aspect, the present invention provides a method for usingGPR49/LGR5, and/or the factors of the Hedgehog pathway such as Sonichedgehog (SHH), PTCH1, GLI1, and GLI2, and/or the factors of the Wntpathway such as LRP6 and β-catenin as an index for identifying a cellhaving a high proliferation ability among corneal endothelial cellsand/or the differentiation ability of a corneal endothelial cell, or amethod for detecting or diagnosing a cell having a high proliferationability among corneal endothelial cells and/or the differentiationability of a corneal endothelial cell.

The method of the present invention can be implemented by performing,for example, a step of detecting GPR49/LGR5, and/or the factors of theHedgehog pathway such as SHH, PTCH1, GLI1, and GLI2, and/or the factorsof the Wnt pathway such as LRP6 and β-catenin, or genes of these factorsa living body, for using GPR49/LGR5, and/or the factors of the Hedgehogpathway such as SHH, PTCH1, GLI1, and GLI2, and/or the factors of theWnt pathway such as LRP6 and β-catenin as an index for identifying acell having a high proliferation ability among corneal endothelial cellsand/or the differentiation ability of a corneal endothelial cell. Forexample, in such a case, a detection agent comprising a substancebinding to GPR49/LGR5, and/or the factors of the Hedgehog pathway suchas Sonic hedgehog (SHH), PTCH1, GLI1, and GLI2, and/or the factors ofthe Wnt pathway such as LRP6 and β-catenin, or genes of these factorscan be used. Such a detection agent is described herein, and it isunderstood that a person skilled in the art can implement the method ofthe present invention based on the description.

In the method of the present invention, the detection agent or thediagnostic agent of the present invention is contacted with an objectivesample, and whether there are GPR49/LGR5, and/or the factors of theHedgehog pathway such as Sonic hedgehog (SHH), PTCH1, GLI1, and GLI2,and/or the factors of the Wnt pathway such as LRP6 and β-catenin, orgenes of these factors, being an objective subject, in the sample isdetermined, or the level or amount thereof is measured.

“Contact (contacted)”, as used herein, means that a substance is made tophysically approach a polypeptide or a polynucleotide which can functionas the marker, the detection agent, the diagnostic, the ligand or thelike of the present invention, either directly or indirectly. Thepolypeptide or the polynucleotide can be made to exist in many buffers,salts, solutions or the like. Contact includes placing a compound on,for example, a beaker, a microtiter plate, a cell culturing flask or amicroarray (e.g., gene chip), containing a polypeptide encoding anucleic acid molecule or a fragment thereof.

In one embodiment, a cell having a high proliferation ability which is asubject in the method of the present invention is an undifferentiatedcell.

In another embodiment, a cell having a high proliferation ability whichis a subject in the method of the present invention is a stem cell.

In another aspect, a corneal endothelial cell which is a subject in themethod of the present invention is a human cell.

In still another embodiment, the proliferation ability of a cornealendothelial cell which is a subject in the method of the presentinvention is identified by a characteristic selected from the groupconsisting of colony forming ability, Ki-67 positivity and BrdUpositivity.

In one embodiment of the present invention, diagnosis regarding thedifferentiated state can be performed, based on the method which is anindex of the present invention.

A specific method for detecting expression of GPR49/LGR5, and/or thefactors of the Hedgehog pathway such as SHH, PTCH1, GLI1, and GLI2,and/or the factors of the Wnt pathway such as LRP6 and β-catenin, orgenes of these factors is not particularly limited, as far as it is amethod which can detect expression of GPR49/LGR5, and/or the factors ofthe Hedgehog pathway such as SHH, PTCH1, GLI1, and GLI2, and/or thefactors of the Wnt pathway such as LRP6 and β-catenin, or genes of thesefactors, in a test sample (e.g., cell), and includes a hybridizationmethod, a nucleic acid amplification method, and an antigen-antibodyreaction method.

Herein, a “test sample” may be a sample that is a corneal endothelialcell or an objective cell, or a substance derived therefrom, which isthought to contain a matter that enables gene expression. For example, acell which is directly isolated from the corneal endothelium can beused. A cell of the corneal endothelium can be obtained by a knownmethod (Koizumi N, Okumura N, Kinoshita S., Experimental Eye Research.2012; 95: 60-7.). Preferably, a cell obtained from a donor of thecorneal endothelium, a corneal endothelial cell or the like can be usedas a test cell sample. Alternatively, a cultured cell containing acorneal endothelial cell which was differentiation-induced in vitro canbe used as a sample. In vitro differentiation induction into a cornealendothelial cell can be implemented by performing differentiationtreatment using a known cell such as an ES cell, an iPS cell, and a bonemarrow parenchymal cell as a starting material by a known method, forexample, an AMED method <see Ueno M, Matsumura M, Watanabe K, NakamuraT, Osakada F, Takahashi M, Kawasaki H, Kinoshita S, Sasai Y; Proc NatlAcad Sci USA. 103 (25) : 9554-9559, 2006.>.

According to one embodiment of detection in accordance with the presentinvention, expression of GPR49/LGR5 in a cell sample can be detected byhybridizing the probe in accordance with the present invention with anucleic acid sample (mRNA or a transcription product thereof), anddirectly or indirectly detecting a hybridization complex, that is, adouble-stranded nucleotide. Regarding a detailed procedure of ahybridization method, see “Molecular Cloning, A Laboratory Manual 2^(nd)ed.” (Cold Spring Harbor Press (1989), particularly Section 9.47-9.58),“Current Protocols in Molecular Biology” (John Wiley & Sons (1987-1997),particularly Section 6.3-6.4), and “DNA Cloning 1: Core Techniques, APractical Approach 2^(nd) ed.” (Oxford University (1995), regarding thecondition, see particularly section 2.10).

Detection of expression of GPR49/LGR5, and/or the factors of theHedgehog pathway such as SHH, PTCH1, GLI1, and GLI2, and/or the factorsof the Wnt pathway such as LRP6 and β-catenin, or genes of these factorsutilizing a hybridization method can be implemented by, for example, (a)a step of contacting a polynucleotide derived from a test sample withthe probe in accordance with the present invention; and (b) a step ofdetecting a hybridization complex. In the step (a), mRNA prepared froman objective test sample or complementary DNA (cDNA) transcribed fromthe mRNA as a polynucleotide derived from a test cell sample can becontacted with the probe. In a detection method using a probe, the probecan be labeled before use. Examples of the label include labelsutilizing radioactivity (e.g., ³²P, ¹⁴C, and ³⁵S), fluorescence (e.g.,FITC and europium), or an enzyme reaction such as chemiluminescence(e.g., peroxidase and alkaline phosphatase). Detection of ahybridization product can be performed using a well-known method such asNorthern hybridization, Southern hybridization, or colony hybridization.Since a cell from which a hybridization complex was detected is a cellexpressing GPR49/LGR5, and/or the factors of the Hedgehog pathway suchas SHH, PTCH1, GLI1, and GLI2, and/or the factors of the Wnt pathwaysuch as LRP6 and β-catenin, the cell can be determined to have a highproliferation ability (an undifferentiated cell, a precursor cell or astem cell) and/or a high differentiation ability.

According to another embodiment of detection in accordance with thepresent invention, expression of GPR49/LGR5, and/or the factors of theHedgehog pathway such as SHH, PTCH1, GLI1, and GLI2, and/or the factorsof the Wnt pathway such as LRP6 and β-catenin, or genes of these factorsin a sample can be detected by amplifying a nucleic acid sample (mRNA ora transcription product thereof) by a nucleic acid amplification methodusing the primer or the primer set in accordance with the presentinvention, and detecting the amplification product.

Detection of expression of GPR49/LGR5, and/or the factors of theHedgehog pathway such as SHH, PTCH1, GLI1, and GLI2, and/or the factorsof the Wnt pathway such as LRP6 and β-catenin, or genes of these factorsutilizing a nucleic acid amplification method can be implemented, forexample, by (i) a step of implementing a nucleic acid amplificationmethod using the primer or the primer set in accordance with the presentinvention and employing a polynucleotide derived from a test sample as atemplate; and (ii) a step of detecting the formed amplification product.

In the step (i), mRNA prepared from an objective test sample orcomplementary DNA (cDNA) transcribed from the mRNA can be used as atemplate. Detection of an amplification product can be implemented usinga nucleic acid amplification method such as a PCR method, an RT-PCRmethod, a real time PCR method, or a LAMP method. Since a cell fromwhich an amplification product is detected is a cell expressingGPR49/LGR5, the cell can be determined to have a high proliferationability (an undifferentiated cell, a precursor cell or a stem cell)and/or a high differentiation ability.

According to another embodiment of detection in accordance with thepresent invention, expression of GPR49/LGR5, and/or the factors of theHedgehog pathway such as SHH, PTCH1, GLI1, and GLI2, and/or the factorsof the Wnt pathway such as LRP6 and β-catenin in a sample can bedetected by contacting the antibody in accordance with the presentinvention with a sample, and detecting an antigen-antibody reaction.

Detection of expression of GPR49/LGR5, and/or the factors of theHedgehog pathway such as SHH, PTCH1, GLI1, and GLI2, and/or the factorsof the Wnt pathway such as LRP6 and β-catenin utilizing anantigen-antibody reaction can be implemented, for example, by thefollowing steps: (I) a step of contacting a protein derived from a testcell sample with the antibody in accordance with the present invention;and (II) a step of measuring an antigen-antibody complex. A method fordetecting an antigen-antibody reaction is well-known to a person skilledin the art, and for example, GPR49/LGR5 protein, and/or the factors ofthe Hedgehog pathway such as SHH, PTCH1, GLI1, and GLI2, and/or thefactors of the Wnt pathway such as LRP6 and β-catenin in a test cellsample which is thought to contain a corneal endothelial cell can bedetected by an immunological method. As the immunological method,concerning a sample obtained by, if necessary, subjecting a cell sampleto an appropriate treatment, for example, separation of a cell or anextraction operation, a known method such as an immunohistologicalstaining method, an enzyme immunometric assay, a Western blottingmethod, an agglutination method, a competition method, or a sandwichmethod can be applied. The immunohistological staining method can beperformed, for example, by a direct method using a labeled antibody, anindirect method using a labeled antibody to the above antibody, or thelike. As a labeling agent, a known labeling substance such as afluorescent substance, a radioactive substance, an enzyme, a metal, or adye can be used.

Since a cell from which an antigen-antibody complex is detected is acell expressing GPR49/LGR5, and/or the factors of the Hedgehog pathwaysuch as SHH, PTCH1, GLI1, and GLI2, and/or the factors of the Wntpathway such as LRP6 and β-catenin, the cell can be determined to have ahigh proliferation ability (an undifferentiated cell, a precursor cellor a stem cell) and/or a high differentiation ability. For use intreatment of a disease requiring transplantation of the cornealendothelium, for example, bullous keratopathy, corneal edema, leukomacorneae, particularly, corneal dystrophy, and a corneal endothelialdisorder caused by trauma or internal eye operation, or other specifiedcorneal endothelial diseases (Fuchs endothelial corneal dystrophy, backpolymorphic endothelial corneal dystrophy etc.), it is desirable that acell having a high proliferation ability (an undifferentiated cell, aprecursor cell or a stem cell) has a high purity.

By performing the above-mentioned respective detection steps not onlyonce, but also by repeating or combining the same steps, the precisionof detection or selection of a cell having a high proliferationability/differentiation ability can be enhanced. Therefore, when such anembodiment is adopted, according to the detection method in accordancewith the present invention, a cell having a high proliferationability/differentiation ability can be detected or selected moreprecisely, by performing the above-mentioned steps two or more times.

In addition, the precision of detection or selection of a cell having ahigh proliferation ability/differentiation ability can be enhanced byconcurrently using another marker gene, preferably, a proliferationmarker gene (e.g., Ki-67 and BrdU) other than genes encoding GPR49/LGR5,and/or the factors of the Hedgehog pathway such as SHH, PTCH1, GLI1, andGLI2, and/or the factors of the Wnt pathway such as LRP6 and β-catenin,or these factors.

As used herein, “diagnosis” refers to determination of the currentstatus or the future of a disease, a disorder or a condition in asubject by identifying a variety of parameters associated with such adisease, disorder or condition. By using the method, the apparatus orthe system of the present invention, the state in a body can beexamined, and such information can be used to select a variety ofparameters such as a disease, a disorder, or a condition in a subject,and a formulation or a method for treatment or prevention to beadministered. As used herein, in a narrow sense, the “diagnosis” refersto diagnosis of the current status, and in a broad sense, it includes“early diagnosis”, “presumptive diagnosis”, and “advance diagnosis”.Since the diagnosis method of the present invention, in principle, canutilize what has moved out of a body, and can be implementedindependently of healthcare professionals such as a doctor, it isindustrially useful. As used herein, in order to make clear that thediagnosis method can be implemented independently of healthcareprofessionals such as a doctor, particularly, “presumptive diagnosis,advance diagnosis or diagnosis” may be named as “assistance”.

A procedure of formulating a diagnostic agent or the like of the presentinvention as a pharmaceutical or the like is known in the art, and isdescribed, for example, in Japanese Pharmacopoeia, U.S. Pharmacopoeia,and other countries' Pharmacopoeias. Therefore, a person skilled in theart can determine the amount of the diagnostic agent to be used with thedescriptions described herein without performing undue experiments.

An antibody used in the present invention can be produced as follows.

An antibody used in the present invention (e.g., anti-GPR49/LGR5antibody, an antibody to the factors of the Hedgehog pathway such asSHH, PTCH1, GLI1, and GLI2, and an antibody to the factors of the Wntpathway such as LRP6 and β-catenin) can be obtained as a polyclonal ormonoclonal antibody using known means. As an antibody used in thepresent invention, particularly, a monoclonal antibody derived from amammal is preferable. Monoclonal antibodies derived from a mammalinclude a monoclonal antibody produced by a hybridoma, and a monoclonalantibody produced by a host transformed with an expression vectorcomprising an antibody gene by a genetic engineering procedure.

As one example, a method for preparing a monoclonal antibody will bedescribed below. The monoclonal antibody can be prepared by preparing ahybridoma by cell fusion between an antibody producing cell obtainedfrom an animal immunized with an antigen and a myeloma cell, andselecting a clone producing an antibody which specifically inhibits theactivity of GPR49/LGR5, R-spondins, the factors of the Hedgehog pathwaysuch as SHH, PTCH1, GLI1, and GLI2, and/or the factors of the Wntpathway such as LRP6 and β-catenin from the resulting hybridoma.

The whole of an amino acid sequence of a protein such as a matureprotein, such as GPR49/LGR5, R-spondins, the factors of the Hedgehogpathway such as SHH, PTCH1, GLI1, and GLI2, and/or the factors of theWnt pathway such as LRP6 and β-catenin used as an antigen inimmunization of an animal, or a fragment thereof having immunogenicitycan be used as an immunogen. In addition, it is preferable to use apeptide consisting of any 10 or more in an amino acid sequence of aprotein of the marker of the present invention as an antigen, as amonoclonal antibody for specifically detecting a protein existing on acell surface. Concerning any of the other factors of the presentinvention (e.g., GPR49/LGR5, R-spondins, the factors of the Hedgehogpathway such as SHH, PTCH1, GLI1, and GLI2, and/or the factors of theWnt pathway such as LRP6 and β-catenin as well as proteins correspondingto them), an antigen can be designed similarly.

After binding to the resulting GPR49/LGR5, R-spondins, the factors ofthe Hedgehog pathway such as SHH, PTCH1, GLI1, and GLI2, and/or thefactors of the Wnt pathway such as LRP6 and β-catenin for an antigen, anadjuvant is added. The adjuvant includes a Freund complete adjuvant anda Freund incomplete adjuvant, and any of them may be mixed.

The antigen obtained as described above is administered to a mammal, forexample, a mouse, a rat, a horse, a monkey, a rabbit, a goat, or sheep.Any method can be used for immunization as far as it is an existingmethod, and immunization is mainly performed by intravenous injection,subcutaneous injection, intraperitoneal injection or the like. Inaddition, an interval of immunization is not particularly limited, andimmunization is performed at an interval of a few days to a few weeks,preferably at an interval of 4 to 21 days.

After 2 to 3 days from the day of last immunization, an antibodyproducing cell is collected. Examples of the antibody producing cellinclude a spleen cell, a lymph node cell, and a peripheral blood cell,and a spleen cell is generally used. As the immunization amount of anantigen, for example, 100 μg of an antigen is used once per mouse.

A monoclonal antibody producing hybridoma can be basically produced asfollows using a known technique. First, an objective protein (e.g., aprotein such as GPR49/LGR5, the factors of the Hedgehog pathway such asSHH, PTCH1, GLI1, and GLI2, and/or the factors of the Wnt pathway suchas LRP6 and β-catenin) as a sensitizing antigen is immunized accordingto a normal immunization method. An immune cell obtained from animmunized animal is fused with a known parent cell by a normal cellfusion method to obtain a hybridoma. Further, by screening a cellproducing an objective antibody from this hybridoma by a normalscreening method, a hybridoma producing an objective antibody can beselected.

Specifically, production of the monoclonal antibody is performed, forexample, as shown below. First, by expressing an objective gene (aGPR49/LGR5 gene, the factors of the Hedgehog pathway such as SHH, PTCH1,GLI1, and GLI2, and/or the factors of the Wnt pathway such as LRP6 andβ-catenin), an objective protein used as a sensitizing antigen forobtaining an antibody can be obtained. A nucleotide sequence of theobjective gene is described in other places of the present description(e.g., disclosed in NCBI registration number NM_003667.2 (SEQ ID No.: 1)and NM_000193 (SEQ ID No.: 11)). That is, after a gene sequence encodingthe objective gene is inserted into a known expression vector totransform an appropriate host cell, an objective protein can be purifiedfrom the host cell or the culture supernatant by a known method.Alternatively, a purified natural protein can also be used similarly.Alternatively, as used in the present invention, a fusion proteinobtained by fusing a desired partial polypeptide of an objective proteinwith a different polypeptide can also be utilized as an immunogen. Inorder to produce the fusion protein as an immunogen, for example, a Fcfragment of an antibody or a peptide tag can be utilized. A vectorexpressing the fusion protein can be produced by fusing genes encodingdesired two or more kinds of polypeptide fragments in frame, andinserting the fused gene in an expression vector. A method of producingthe fusion protein is described in Molecular Cloning 2nd ed. (Sambrook,J et al., Molecular Cloning 2nd ed., 9.47-9.58, Cold Spring Harbor Lab.press, 1989).

The thus purified objective protein (e.g., a protein such as GPR49/LGR5,the factors of the Hedgehog pathway such as SHH, PTCH1, GLI1, and GLI2,and/or the factors of the Wnt pathway such as LRP6 and β-catenin) can beused as a sensitizing antigen used in immunization of a mammal. Apartial peptide of an objective protein can also be used as asensitizing antigen. For example, the following peptides can be used asa sensitizing antigen: a peptide obtained by chemical synthesis based onan amino acid sequence of an objective protein (a protein such as humanGPR49/LGR5, the factors of the Hedgehog pathway such as SHH, PTCH1,GLI1, and GLI2, and/or the factors of the Wnt pathway such as LRP6 andβ-catenin); a peptide obtained by incorporating a part of an objectivegene (a gene encoding human GPR49/LGR5, the factors of the Hedgehogpathway such as SHH, PTCH1, GLI1, and GLI2, and/or the factors of theWnt pathway such as LRP6 and β-catenin) into an expression vector toexpress the gene; and a peptide obtained by degrading an objectiveprotein (a protein such as human GPR49/LGR5, the factors of the Hedgehogpathway such as SHH, PTCH1, GLI1, and GLI2, and/or the factors of theWnt pathway such as LRP6 and β-catenin) with a protease.

The region and size of a protein used as a partial peptide are notlimited. As an example of the region, in the case of GPR49/LGR5, aregion can be selected from an amino acid sequence constituting anextracellular domain of GPR49/LGR5 (1-556^(th), 615-637^(th),704-722^(nd), and 792-800^(th) amino acids in an amino acid sequence ofSEQ ID No.: 2). It is preferable that the number of amino acidsconstituting a peptide used as a sensitizing antigen is at least 3, forexample, 5 or more, or 6 or more. More specifically, a peptide of 8 to50, preferably 10 to 30 residues can be used as a sensitizing antigen.Also in the case of the factors of the Hedgehog pathway such as SHH,PTCH1, GLI1, and GLI2, and/or the factors of the Wnt pathway such asLRP6 and β-catenin, an appropriate peptide can be used as a sensitizingantigen similarly based on information such as sequences describedherein, for example, SEQ ID Nos.: 12, 44, 46, 48, 50, and 52.

A mammal to be immunized with the sensitizing antigen is notparticularly limited. In order to obtain the monoclonal antibody by acell fusion method, it is preferable to select an immune animal in viewof compatibility with a parent cell to be used in cell fusion.Generally, rodent animals are preferable as an immune animal.Specifically, a mouse, a rat, a hamster or a rabbit can be used as animmune animal. In addition, a monkey and the like can also be used as animmune animal.

The animals can be immunized with a sensitizing antigen according to aknown method. For example, as a general method, a mammal can beimmunized by injecting a sensitizing antigen intraperitoneally orsubcutaneously. Specifically, the sensitizing antigen is administered toa mammal several times every 4 to 21 days. The sensitizing antigen isdiluted with PBS (Phosphate-Buffered Saline) or physiological saline atan appropriate dilution rate, and is used in immunization. Furthermore,the sensitizing antigen can be administered together with an adjuvant.For example, the sensitizing antigen can be mixed with a Freund completeadjuvant, and emulsified to obtain a sensitizing antigen. In addition,in immunization with the sensitizing antigen, an appropriate carrier canbe used. Particularly, when a partial peptide having a small molecularweight is used as the sensitizing antigen, it is desirable to bind thesensitizing antigen peptide with a carrier protein such as albumin orkeyhole limpet hemocyanin to perform immunization.

In addition, the monoclonal antibody can also be obtained by DNAimmunization. DNA immunization is a method for imparting immunestimulation by administering vector DNA constructed in such a form thata gene encoding an antigenic protein can be expressed in an immuneanimal to the immune animal, and expressing an immunizing antigen in aliving body of the immune animal. In order to obtain the monoclonalantibody of the present invention by DNA immunization, first, DNAexpressing an objective protein (e.g., GPR49/LGR5, and/or the factors ofthe Hedgehog pathway such as SHH, PTCH1, GLI1, and GLI2, and/or thefactors of the Wnt pathway such as LRP6 and β-catenin) is administeredto the immune animal. DNA encoding an objective protein (e.g.,GPR49/LGR5, the factors of the Hedgehog pathway such as SHH, PTCH1,GLI1, and GLI2, and/or the factors of the Wnt pathway such as LRP6 andβ-catenin) can be synthesized by a known method such as PCR. Theresulting DNA is inserted into an appropriate expression vector, and isadministered to the immune animal. As the expression vector, forexample, a commercially available expression vector such as pcDNA3.1 canbe utilized. As a method of administering the vector to a living body, agenerally used method can be utilized. For example, by shooting goldparticles with an expression vector adsorbed thereon into a cell with agene gun, DNA immunization can be performed.

After a mammal is immunized in this way, and an increase in the amountof the desired antibody in serum is confirmed, an immune cell iscollected from the mammal, and is subjected to cell fusion. As apreferable immune cell, particularly, a spleen cell can be used.

As a cell to be fused with the immune cell, a myeloma cell of a mammalis used. It is preferable that the myeloma cell has an appropriateselection marker for screening. The selection marker refers to acharacter that a cell can survive (or cannot survive) under specifiedculturing conditions. As the selection marker,hypoxanthine-guanine-phosphoribosyl transferase deficiency (hereinafter,abbreviated as HGPRT deficiency) or thymidine kinase deficiency(hereinafter, abbreviated as TK deficiency) is known. A cell havingdeficiency of HGPRT or TK has hypoxanthine-aminopterin-thymidinesensitivity (hereinafter, abbreviated as HAT sensitivity). AHAT-sensitive cell cannot synthesize DNA in a HAT selective medium, anddies. However, when the cell is fused with a normal cell, it comes to beable to proliferate even in the HAT selective medium since the cell cancontinue synthesis of DNA utilizing a salvage circuit of a normal cell.

A HGPRT-deficient or TK-deficient cell can be selected in a mediumcontaining 6-thioguanine, 8-azaguanine (hereinafter, abbreviated as8AG), or 5′-bromodeoxyuridine. Since a normal cell takes thesepyrimidine analogs into DNA, it dies. However, a cell deficient in theseenzymes cannot take in these pyrimidine analogs, it can survive in aselective medium. In addition, a selection marker called G418 resistanceimparts resistance to a 2-deoxystreptamine antibiotic (gentamycinanalog) by a neomycin resistance gene. A variety of myeloma cellssuitable for cell fusion are known. Basically, cell fusion between animmune cell and a myeloma cell is performed in accordance with a knownmethod, for example, Koehler C. and Milstein C., Methods Enzymol. (1981)73, 3-46. More specifically, for example, cell fusion can be carried outin a normal nutrient culture solution in the presence of a cell fusionpromoter. As the fusion promoter, for example, polyethylene glycol (PEG)or a sendaivirus (HVJ) can be employed. Further, in order to enhance thefusion efficiency, an assistant such as dimethyl sulfoxide can beoptionally added. The use ratio between an immune cell and a myelomacell can be arbitrarily set. For example, it is preferable that 1 to 10times of the immune cells are used relative to the myeloma cells. As aculture solution used in cell fusion, for example, an RPMI1640 culturesolution suitable for proliferation of a myeloma cell strain, an MEMculture solution, and a normal culture solution used in this kind ofcell culturing can be utilized. Further, a serum replenisher solutionsuch as fetal calf serum (FCS) can be utilized.

In cell fusion, by mixing predetermined amounts of the immune cell andthe myeloma cell in a culture solution well, and mixing a PEG solutionwhich has been warmed to around 37° C. in advance, an objective fusedcell (hybridoma) is formed. In a cell fusion method, for example, PEGhaving an average molecular weight of around 1000 to 6000 can be addedusually in a concentration of 30% (w/v) to 60% (w/v). Subsequently, byrepeating an operation of sequentially adding suitable culture solutionslisted above, and centrifuging the resultant to remove the supernatant,a cell fusion agent and the like which are not preferable for the growthof a hybridoma are removed.

The thus obtained hybridoma can be selected by utilizing a selectionculture solution appropriate for a selection marker possessed by amyeloma used in cell fusion. For example, a cell having deficiency ofHGPRT or TK can be selected by culturing in a HAT culture solution (aculture solution containing hypoxanthine, aminopterin and thymidine).That is, when a HAT-sensitive myeloma cell is used in cell fusion, acell succeeded in fusion with a normal cell can be selectivelyproliferated in a HAT culture solution. Culturing using the HAT culturesolution is continued for a period sufficient for a cell other than anobjective hybridoma (non-fused cell) to die. Specifically, generally,the objective hybridoma can be selected by culturing for a few days to afew weeks. Then, by performing a normal limiting dilution method,screening and single cloning of a hybridoma producing an objectiveantibody can be performed. Alternatively, an antibody recognizing anobjective protein (e.g., GPR49/LGR5, the factors of the Hedgehog pathwaysuch as SHH, PTCH1, GLI1, and GLI2, and/or the factors of the Wntpathway such as LRP6 and β-catenin) can be produced by the methoddescribed in International Publication WO 03/104453.

Screening and single cloning of an objective antibody can beappropriately performed by a screening method based on a knownantigen-antibody reaction. For example, an antigen is bound to a carriersuch as beads made of polystyrene or the like, or a commerciallyavailable 96-well microtiter plate, and the resultant is reacted withthe culture supernatant of a hybridoma. Then, after the carrier iswashed, the carrier is reacted with a secondary antibody or the likelabeled with an enzyme. When an objective antibody reacting with asensitizing antigen is contained in the culture supernatant, thesecondary antibody binds to the carrier via this antibody. By finallydetecting the secondary antibody binding to the carrier, whether theobjective antibody exists in the culture supernatant or not can bedetermined. It becomes possible to clone a hybridoma producing a desiredantibody having an ability to bind to an antigen by a limiting dilutionmethod or the like. In this case, as an antigen, a protein such asGPR49/LGR5, the factors of the Hedgehog pathway such as SHH, PTCH1,GLI1, and GLI2, and/or the factors of the Wnt pathway such as LRP6 andβ-catenin, which is substantially of the same nature, including thoseused in immunization can be preferably used. For example, a cell strainexpressing an objective protein (e.g., GPR49/LGR5, the factors of theHedgehog pathway such as SHH, PTCH1, GLI1, and GLI2, and/or the factorsof the Wnt pathway such as LRP6 and β-catenin), an extracellular domainof an objective protein (e.g., GPR49/LGR5, the factors of the Hedgehogpathway such as SHH, PTCH1, GLI1, and GLI2, and/or the factors of theWnt pathway such as LRP6 and β-catenin), or an oligopeptide consistingof a partial amino acid sequence constituting the region can be utilizedas an antigen.

In addition, an objective antibody can also be obtained byantigen-sensitizing a human lymphocyte, in addition to a method ofobtaining the hybridoma by immunizing an animal other than human with anantigen. Specifically, first, a human lymphocyte is in vitro sensitizedwith a protein such as GPR49/LGR5, the factors of the Hedgehog pathwaysuch as SHH, PTCH1, GLI1, and GLI2, and/or the factors of the Wntpathway such as LRP6 and β-catenin. Then, an immunized lymphocyte isfused with an appropriate fusion partner. As the fusion partner, forexample, a myeloma cell which is derived from human and has a permanentdivision potential can be utilized (see JP-H01-59878 B(Kokoku)). Ananti-GPR49/LGR5 antibody obtained by this method is a human antibodyhaving an activity of binding to a GPR49/LGR5 protein. This also appliesto the factors of the Hedgehog pathway such as SHH, Ptch1, Gli1, andGli2, and/or the factors of the Wnt pathway such as LRP6 and β-catenin.

Further, by administering an objective protein (e.g., a protein such asGPR49/LGR5, the factors of the Hedgehog pathway such as SHH, PTCH1,GLI1, and GLI2, and/or the factors of the Wnt pathway such as LRP6 andβ-catenin) which is to be an antigen to a transgenic animal having thewhole repertory of a human antibody gene, or immunizing the animal withDNA which was constructed so as to express an objective protein (e.g.,GPR49/LGR5) in the animal, a human antibody to an objective protein(e.g., GPR49/LGR5, the factors of the Hedgehog pathway such as SHH,PTCH1, GLI1, and GLI2, and/or the factors of the Wnt pathway such asLRP6 and β-catenin) can be obtained. An antibody producing cell of animmune animal can be immortalized by cell fusion with an appropriatefusion partner or treatment such as infection with an Epstein-Barrvirus. From the thus obtained immortalized cell, a human antibody to anobjective protein (e.g., GPR49/LGR5, the factors of the Hedgehog pathwaysuch as SHH, PTCH1, GLI1, and GLI2, and/or the factors of the Wntpathway such as LRP6 and β-catenin) can be isolated (see InternationalPublications WO 94/25585, WO 93/12227, WO 92/03918, and WO 94/02602).Further, by cloning the immortalized cell, a cell producing an antibodyhaving objective reaction specificity can be cloned. When a transgenicanimal is an immune animal, an immune system of the animal recognizes anobject human protein (e.g., human GPR49/LGR5) as a foreign substance.Therefore, a human antibody to an objective human protein (e.g., humanGPR49/LGR5, the factors of the Hedgehog pathway such as SHH, PTCH1,GLI1, and GLI2, and/or the factors of the Wnt pathway such as LRP6 andβ-catenin) can be easily obtained.

The thus produced hybridoma producing a monoclonal antibody can besubcultured in a normal culture solution. Alternatively, the hybridomacan also be preserved in liquid nitrogen over a long term. The hybridomais cultured according to a normal method, and from the culturesupernatant, an objective monoclonal antibody can be obtained.Alternatively, a monoclonal antibody can also be obtained as ascites byadministering the hybridoma to a mammal having compatibility therewithand proliferating the hybridoma. The former method is suitable forobtaining an antibody having high purity.

In the present invention, an antibody encoded by an antibody gene whichwas cloned from an antibody producing cell can also be utilized. Byincorporating the cloned antibody gene into an appropriate vector andintroducing it into a host, the gene can be expressed as an antibody.Isolation of the antibody gene, introduction into the vector, and amethod for transforming a host cell have already been established (e.g.,see Vandamme, A. M., et al., Eur. J. Biochem. (1990) 192, 767-775).

For example, for the purpose of obtaining an antibody to an objectiveprotein (e.g., GPR49/LGR5, the factors of the Hedgehog pathway such asSHH, PTCH1, GLI1, and GLI2, and/or the factors of the Wnt pathway suchas LRP6 and β-catenin), it is further preferable that binding of anantibody to an objective protein (e.g., GPR49/LGR5, the factors of theHedgehog pathway such as SHH, PTCH1, GLI1, and GLI2, and/or the factorsof the Wnt pathway such as LRP6 and β-catenin) is specific. An antibodybinding to an objective protein (e.g., GPR49/LGR5, the factors of theHedgehog pathway such as SHH, PTCH1, GLI1, and GLI2, and/or the factorsof the Wnt pathway such as LRP6 and β-catenin) can be screened, forexample, by a method comprising (1) a step of contacting an antibodycomprising a V region encoded by the resulting cDNA with the objectiveprotein (e.g., GPR49/LGR5, the factors of the Hedgehog pathway such asSHH, PTCH1, GLI1, and GLI2, and/or the factors of the Wnt pathway suchas LRP6 and β-catenin); (2) a step of detecting binding between theobjective protein (e.g., GPR49/LGR5, the factors of the Hedgehog pathwaysuch as SHH, PTCH1, GLI1, and GLI2, and/or the factors of the Wntpathway such as LRP6 and β-catenin) and an antibody, and (3) a step ofselecting an antibody binding to the objective protein (e.g.,GPR49/LGR5, the factors of the Hedgehog pathway such as SHH, PTCH1,GLI1, and GLI2, and/or the factors of the Wnt pathway such as LRP6 andβ-catenin).

A method for detecting binding between an antibody and the objectiveprotein (e.g., GPR49/LGR5, the factors of the Hedgehog pathway such asSHH, PTCH1, GLI1, and GLI2, and/or the factors of the Wnt pathway suchas LRP6 and β-catenin) is known. Specifically, a test antibody isreacted with the objective protein (e.g., GPR49/LGR5, the factors of theHedgehog pathway such as SHH, PTCH1, GLI1, and GLI2, and/or the factorsof the Wnt pathway such as LRP6 and β-catenin) immobilized on a carrierand, then, the resultant is reacted with a labeled antibody recognizingan antibody. When a labeled antibody is detected on a carrier afterwashing, binding of the test antibody to the objective protein (e.g.,GPR49/LGR5, the factors of the Hedgehog pathway such as SHH, PTCH1,GLI1, and GLI2, and/or the factors of the Wnt pathway such as LRP6 andβ-catenin) can be demonstrated. For labeling, an enzymatically activeprotein such as peroxidase and β-glactosidase, or a fluorescentsubstance such as FITC can be utilized. For evaluating the bindingactivity of an antibody, a fixed specimen of a cell expressing theobjective protein (e.g., GPR49/LGR5, the factors of the Hedgehog pathwaysuch as SHH, PTCH1, GLI1, and GLI2, and/or the factors of the Wntpathway such as LRP6 and β-catenin) can also be utilized.

As a method for screening an antibody using the binding activity as anindex, a panning method utilizing a phage vector can also be used. Whenan antibody gene is obtained as a library of a subclass of a heavy chainand a light chain as described above, a screening method utilizing aphage vector is advantageous. Genes encoding variable regions of a heavychain and a light chain can be prepared into a single chain Fv (scFv) bylinking with an appropriate linker sequence. When a gene encoding scFvis inserted into a phage vector, a phage expressing scFv on a surfacecan be obtained. When this phage is contacted with an objective antigen,and a phage bound to the antigen is recovered, DNA encoding scFv havingthe objective binding activity can be recovered. By repeating thisoperation, if necessary, scFv having the objective binding activity canbe concentrated.

In the present invention, a polynucleotide encoding an antibody mayencode the full length of an antibody, or may encode a part of anantibody. A part of an antibody refers to any part of an antibodymolecule. Hereinafter, as used herein, an antibody fragment may be usedin implementation of the present invention. A preferable antibodyfragment in the present invention comprises a complementaritydetermination region (CDR) of an antibody. Further preferably, theantibody fragment of the present invention comprises all of three CDRsconstituting a variable region.

In one embodiment, the antibody of the present invention includes notonly a divalent antibody, a representative of which is IgG, but also amonovalent antibody, and a polyvalent antibody, a representative ofwhich is IgM, as far as it binds to the objective protein (e.g.,GPR49/LGR5, the factors of the Hedgehog pathway such as SHH, PTCH1,GLI1, and GLI2, and/or the factors of the Wnt pathway such as LRP6 andβ-catenin). The polyvalent antibody of the present invention includes apolyvalent antibody all having the same antigen-binding site, and apolyvalent antibody having a partially or entirely differentantigen-binding site. The antibody of the present invention is notlimited to a full length molecule of an antibody, and may be alow-molecular antibody or a modification product thereof, as far as itbinds to the objective protein (e.g., GPR49/LGR5, the factors of theHedgehog pathway such as SHH, PTCH1, GLI1, and GLI2, and/or the factorsof the Wnt pathway such as LRP6 and β-catenin).

In order to confirm the immune response level of an immunized animal,and select an objective hybridoma from a cell after cell fusiontreatment, the antibody titer in the blood of an immunized animal, orthe antibody titer in the culture supernatant of an antibody producingcell is measured. Examples of a method for detecting an antibody includea known technique, for example, EIA (enzyme immunoassay), RIA(radioimmunoassay), and ELISA (enzyme linked immunosorbent assay).

According to the immunoassay, even when a sample contains a large amountof contaminating substance, the concentration of a marker can beaccurately measured. Examples of the immunoassay include a classicalmethod such as a precipitation reaction of directly or indirectlymeasuring an antigen-antibody bound product, an agglutination reaction,and a hemolytic reaction, enzyme immunoassay (EIA) whose detectionsensitivity has been enhanced by combining a labeling method therewith,radioimmunoassay (RIA), and fluorescent immunoassay (FIA). In addition,an antibody specific for a marker used in these immunoassays may bemonoclonal or polyclonal.

As a method for ionization when the concentration of a marker ismeasured by mass spectrometry, either of matrix-assisted laserdesorption/ionization (MALDI) and electrospray ionization (ESI) can beapplied, and MALDI producing little polyvalent ions is preferable.Particularly, according to MALDI-TOF-MS which is a combination with atime-of-flight mass spectrometer (TOF), the concentration of a markercan be measured more accurately. Further, according to MS/MS using twomass spectrometers, the concentration of a marker can be measured moreaccurately.

When the concentration of a marker is measured by electrophoresis, forexample, a test material is subjected to SDS-polyacrylamide gelelectrophoresis (SDS-PAGE) to separate an objective marker, the gel isstained with an appropriate dye or fluorescent substance, and theconcentration or fluorescent intensity of a band corresponding to anobjective marker may be measured. When separation of the marker isinsufficient only by SDS-PAGE, two-dimensional electrophoresis which isa combination with isoelectric electrofocusing (IEF) can also be used.Further, not by direct detection from a gel, but by performing Westernblotting, the amount of the marker on a membrane can be measured.

When the concentration of the marker is measured by chromatography, forexample, a method by high performance liquid chromatography (HPLC) canbe used. That is, by subjecting a sample to HPLC to separate anobjective marker, and measuring the peak area of the chromatogram, theconcentration of the marker in a sample can be measured.

As a myeloma cell to be fused with an antibody producing cell, anestablished cell which is derived from a variety of animals such as amouse, a rat, or human, and is generally available to a person skilledin the art is used. As a cell strain to be used, a cell strain which hasdrug resistance, and has such a nature that it cannot survive in aselective medium (e.g., HAT medium) in an unfused state, and can surviveonly in a fused state is used. Generally, an 8-azaguanine-resistantstrain is used, and this cell strain is deficient inhypoxanthine-guanine-phosphoribosyltransferase, and cannot be grown in ahypoxanthine-aminopterin-thymidine (HAT) medium.

As the myeloma cell, already known various cell strains, for example, P3(P3x63Ag8.653) (J. Immunol. (1979) 123: 1548-1550), P3x63Ag8U.1 (CurrentTopics in Microbiology and Immunology (1978) 81: 1-7), NS-1 (Kohler, G.and Milstein, C., Eur. J. Immunol. (1976) 6: 511-519), MPC-11 (MarguliesD. H. et al., Cell (1976) 8: 405-415), SP2/0 (Shulman M. et al., Nature(1978) 276: 269-270), FO (Lazekas de St. Groth, S. and Scheidegger, D.,J. Immunol. Methods (1980) 35: 1-21), S194 (Trowbridge, I. S., J. Exp.Med. (1978) 148: 313-323), and R210 (Galfre G. et al., Nature (1979)277: 131-133) are preferably employed.

An antibody producing cell is obtained from a spleen cell, a lymph nodecell or the like. That is, a spleen, a lymph node or the like isisolated or collected from the animals, and these tissues are crushed.The resulting crushed product is suspended in a medium or a buffer suchas PBS, DMEM, and RPMI1640, and the suspension is filtered with astainless mesh or the like, and centrifuged, thereby, an objectiveantibody producing cell is prepared.

Then, the myeloma cell and the antibody producing cell are fused. Cellfusion is performed by contacting the myeloma cell with the antibodyproducing cell at a mixing ratio of 1:1 to 1:10 at 30 to 37° C. for 1 to15 minutes in a medium for culturing an animal cell such as MEM, DMEM,and RPME-1640 media in the presence of a fusion promoter. In order topromote cell fusion, a fusion promoter or a fusion virus such aspolyethylene glycol having an average molecular weight of 1,000 to6,000, polyvinyl alcohol or a sendaivirus can be used. Alternatively,the antibody producing cell can be fused with the myeloma cell using acommercially available cell fusion apparatus utilizing electricstimulation (e.g., electroporation).

From the cells after cell fusion treatment, an objective hybridoma isselected. Examples of a method for selection include a method thatemploys selective proliferation of a cell in a selective medium. Thatis, a cell suspension is diluted with an appropriate medium, and seededon a microtiter plate, a selective medium (HAT medium etc.) is added toeach well, and thereafter, the selective medium is appropriatelyexchanged to perform culturing. As a result, a growing cell can beobtained as a hybridoma.

Screening of the hybridoma is performed by a limiting dilution method, afluorescence excitation cell sorter method or the like, and finally, amonoclonal antibody-producing hybridoma is obtained. Examples of amethod for collecting a monoclonal antibody from the obtained hybridomainclude a normal cell culturing method and an ascites forming method. Inthe cell culturing method, the hybridoma is cultured in an animal cellculturing medium such as an RPMI-1640 medium containing 10 to 20% fetalcalf serum, a MEM medium, or a serum-free medium for 2 to 14 days undernormal culturing conditions (e.g., 37° C., 5% CO₂ concentration), and anantibody is obtained from the culture supernatant. In the ascitesforming method, the hybridoma is administered into the abdominal cavityof an animal of the same species as a mammal from which the myeloma cellis derived, and the hybridoma is proliferated in a large amount. Then,after 1 to 4 weeks, ascites or serum is collected.

In the method for collecting an antibody, when purification of anantibody is required, the antibody is purified by appropriatelyselecting a known method such as an ammonium sulfate salting-out method,ion exchange chromatography, and affinity chromatography, or combiningthem.

Therefore, a substance contained as an antigen in antibody productionmay be a partial sequence, as far as it comprises at least one epitopewhich can induce immunization, although the full length of a marker as asubject is preferable. An epitope can be used even if the preciseposition and the precise structure thereof have not been necessarilyfound, but if necessary, identification of an epitope in a predeterminedprotein can be easily attained using a technique well-known in the art.For example, Geysen et al. (1984) Proc. Natl. Acad. Sci. USA 81: 3998;U.S. Pat. No. 4,708,871; and Geysen et al. (1986) Molecular Immunology23: 709 can be referred to. Antibodies recognizing the same epitope canbe identified by a simple immunoassay. In this way, a method fordetermining an epitope including a peptide is well-known in the art, andsuch an epitope, when a primary sequence of a nucleic acid or an aminoacid is provided, can be determined by a person skilled in the art usingsuch a well-known conventional technique.

Therefore, for use as an epitope including a peptide, a sequence of alength of at least 3 amino acids is necessary, and preferably, asequence of a length of at least 4 amino acids, more preferably at least5 amino acids, at least 6 amino acids, at least 7 amino acids, at least8 amino acids, at least 9 amino acids, at least 10 amino acids, at least15 amino acids, at least 20 amino acids, or at least 25 amino acids maybe required. The epitope may be straight, or may be in a conformationform.

In one aspect, according to the present invention, there is provided adetection kit for implementing the detection method in accordance withthe present invention.

In one embodiment, the detection kit in accordance with the presentinvention includes a detection kit for implementing detection of anembodiment in accordance with the present invention, specifically, a kitfor detecting expression of GPR49/LGR5, and/or the factors of theHedgehog pathway such as SHH, PTCH1, GLI1, and GLI2, and/or the factorsof the Wnt pathway such as LRP6 and β-catenin, comprising at least theprobe in accordance with the present invention. This probe may belabeled. This kit for detection detects expression of GPR49/LGR5, and/orthe factors of the Hedgehog pathway such as SHH, PTCH1, GLI1, and GLI2,and/or the factors of the Wnt pathway such as LRP6 and β-catenin by ahybrid forming method. Therefore, a detection method of a first aspectcan optionally further comprise a variety of reagents for carrying outthe hybrid forming method, for example, a substrate compound used indetection of a label, a hybridization buffer, a direction, and/or aninstrument.

The detection kit of this embodiment in accordance with the presentinvention may further comprise a probe, a primer, a primer set, or anantibody which can detect expression of a differentiation marker gene(e.g., Ki-67, or BrdU) other than GPR49/LGR5, and/or the factors of theHedgehog pathway such as SHH, PTCH1, GLI1, and GLI2, and/or the factorsof the Wnt pathway such as LRP6 and β-catenin, in order to performdetection precisely. These probe, primer, primer set and antibody may belabeled. This kit for detection further detects expression of adifferentiation marker gene other than GPR49/LGR5, and/or the factors ofthe Hedgehog pathway such as SHH, PTCH1, GLI1, and GLI2, and/or thefactors of the Wnt pathway such as LRP6 and β-catenin, by any of ahybrid forming method, a nucleic acid amplification method, and anantigen-antibody reaction method.

In another embodiment, the kit for detection in accordance with thepresent invention includes a detection kit for carrying out detection ofanother embodiment in accordance with the present invention,specifically, a kit for detecting expression of GPR49/LGR5, and/or thefactors of the Hedgehog pathway such as SHH, PTCH1, GLI1, and GLI2,and/or the factors of the Wnt pathway such as LRP6 and β-catenin,comprising at least the primer in accordance with the present inventionor the primer set in accordance with the present invention. This kit fordetection detects expression of GPR49/LGR5, and/or the factors of theHedgehog pathway such as SHH, PTCH1, GLI1, and GLI2, and/or the factorsof the Wnt pathway such as LRP6 and β-catenin, by a nucleic acidamplification method. Therefore, a detection method of a second aspectmay optionally further comprise a variety of reagents for carrying out anucleic acid amplification method, for example, a buffer, an internalstandard which can indicate that PCR can normally progress, a direction,and/or an instrument.

The detection kit of this embodiment in accordance with the presentinvention may further comprise a probe, a primer, a primer set, or anantibody which can detect expression of a differentiation marker geneother than GPR49/LGR5, and /or the factors of the Hedgehog pathway suchas SHH, PTCH1, GLI1, and GLI2, and/or the factors of the Wnt pathwaysuch as LRP6 and β-catenin, in order to perform detection precisely.These probe, primer, primer set, and antibody may be labeled. This kitfor detection further detects expression of a differentiation markerother than GPR49/LGR5, and/or the factors of the Hedgehog pathway suchas SHH, PTCH1, GLI1, and GLI2, and/or the factors of the Wnt pathwaysuch as LRP6 and β-catenin, by any of a hybrid forming method, a nucleicacid amplification method, and an antigen-antibody reaction method.

In a further embodiment, the detection kit in accordance with thepresent invention includes a detection kit for carrying out detection ofa further embodiment in accordance with the present invention,specifically, a kit for detecting a protein of GPR49/LGR5, and/or thefactors of the Hedgehog pathway such as SHH, PTCH1, GLI1, and GLI2,and/or the factors of the Wnt pathway such as LRP6 and β-catenin,comprising at least the antibody in accordance with the presentinvention. This antibody may be labeled. This kit for detection detectsexpression of GPR49/LGR5, and/or the factors of the Hedgehog pathwaysuch as SHH, PTCH1, GLI1, and GLI2, and/or the factors of the Wntpathway such as LRP6 and β-catenin, by detecting an antigen-antibodyreaction. The detection method of this embodiment may optionally furthercomprise a variety of reagents for carrying out an antigen-antibodyreaction, for example, a secondary antibody, a coloring reagent, abuffer, a direction, and/or an instrument used in an ELISA method or thelike.

In this embodiment, the detection kit in accordance with the presentinvention may further comprise a probe, a primer, a primer set, or anantibody which can detect expression of a differentiation marker otherthan GPR49/LGR5, and/or the factors of the Hedgehog pathway such as SHH,PTCH1, GLI1, and GLI2, and/or the factors of the Wnt pathway such asLRP6 and β-catenin, in order to perform detection precisely. Theseprobe, primer, primer set, and antibody may be labeled. This kit fordetection further detects expression of a differentiation marker otherthan GPR49/LGR5, and/or the factors of the Hedgehog pathway such as SHH,Ptch1, Gli1, and Gli2, and/or the factors of the Wnt pathway such asLRP6 and β-catenin, by any of a hybrid forming method, a nucleic acidamplification method, and an antigen-antibody reaction method.

It can be understood that, in these kit, composition or system, a markerin a sample derived from any subject, the factors specificallyinteracting with the marker, or means selectively recognizing the markercan be used, as far as the marker of the present invention (e.g.,GPR49/LGR5, and/or the factors of the Hedgehog pathway such as SHH,PTCH1, GLI1, and GLI2, and/or the factors of the Wnt pathway such asLRP6 and β-catenin) can be identified. Therefore, it is understood thatnot only a factor or means specifically described herein, but also anyequivalent factor or means known in the art can be used.

In one embodiment, the factor used in the present invention is selectedfrom the group consisting of a nucleic acid molecule, a polypeptide, afat, a sugar chain, an organic low molecule and a complex moleculethereof, and preferably, the factor is a protein or a complex molecule(e.g., a glycoprotein or a lipoprotein). Preferably, the factor is anantibody (e.g., a polyclonal antibody or a monoclonal antibody). It ispreferable that such a factor is labeled, or can be labeled. This isbecause diagnosis becomes easy.

In a preferable embodiment of the present invention, means to be used isselected from the group consisting of a mass spectrometry apparatus, anuclear magnetic resonance measuring apparatus, an X-ray analysisapparatus, SPR, chromatography (e.g., HPLC, thin layer chromatography,or gas chromatography), an immunological means (e.g., Western blotting,EIA (enzyme immunoassay), RIA (radioimmunoassay), or ELISA (enzymelinked immunosorbent assay)), a biochemical means (e.g., pIelectrophoresis, Southern blotting, or two-dimensional electrophoresis),an electrophoresis instrument, a chemical analytical instrument, afluorescent two-dimensional differential electrophoresis method(2DE-DIGE), an isotope-coded affinity tag (ICAT), a tandem affinitypurification method (TAP method), a physical means, lasermicrodissection and a combination thereof.

In a preferable embodiment of the present invention, the system or thekit of the present invention further comprises a standard of a marker.It is preferable that such a standard is used in order to confirmwhether means for detecting a marker (a factor specifically interactingwith the marker, or means selectively recognizing the marker) normallyfunctions or not.

In a preferable embodiment, the present invention can further comprisemeans for purifying a sample as a subject. Examples of such apurification means include chromatography. Since the precision ofdiagnosis can be enhanced by purification, the purification means can beused in a preferable embodiment, but it is not essential.

In one embodiment, the factor or the means used in the present inventionhas an ability to quantitate the marker of the present invention. Suchquantitation may be performed by such means or factor that enables aproper calibration curve to be drawn when a standard curve is drawn.Preferable examples thereof include an antibody, mass spectrometry, andchromatography analysis. Therefore, in a certain embodiment, the systemof the present invention further comprises a quantitation means forquantitating a marker.

In one embodiment, a quantitation means comprises a determination meansfor determining whether the marker is within the range of a normal valueor not, by comparing the standard curve with the measured result. Such adetermination means can be realized using a computer.

In one embodiment, the kit or the system of the present inventioncomprises a composition comprising a marker or the factor specificallyinteracting with a marker.

In one aspect, the present invention provides use of a marker in asample derived from a subject, a factor specifically interacting withthe marker, or means selectively recognizing the marker, in productionof a pharmaceutical for presumptively diagnosing, pre-diagnosing oradvancingly diagnosing, predicting, detecting or diagnosing the level ofthe proliferation ability or the differentiated state, or a disease, adisorder or a condition associated therewith. Herein, a sample may beobtained by any means. Usually, when a person in charge other than adoctor is engaged in measurement, the sample may be one that wasobtained by a doctor in some way. Determination of the level of theproliferation ability or the differentiated state, or whether there is apossibility of a disease, a disorder or a condition associated therewithor not, from the measurement result, can be implemented by determiningwhether the result is abnormal or not as compared with each marker, oras compared with a normal value. In the method of the present invention,it is understood that a marker to be used or the like may have any oneor a plurality of characteristics described in other places of thepresent description, as far as they are not contradictory. In thedetection or the diagnosis of present invention, as a method formeasuring the concentration of a marker, a method which is generallyused for quantitating a protein can be used as it is, as far as it is amethod which can specifically measure the concentration of the marker.For example, various immunoassays, mass spectrometry (MS),chromatography, and electrophoresis can be used.

One preferable embodiment in the detection or the diagnosis of thepresent invention is to trap a marker on a carrier, and measure theconcentration of the trapped marker. That is, a substance havingaffinity for the marker is immobilized on a carrier, and the marker istrapped on the carrier via the substance having affinity. According tothe present embodiment, influence of a contaminating substance containedin a sample can be reduced, and the concentration of the marker can bemeasured precisely at higher sensitivity.

In one embodiment, when an immunoassay is used in a method for measuringa marker, it is preferable to use a carrier on which an antibody isimmobilized. Such use enables easy construction of a system of animmunoassay using an antibody immobilized on a carrier as a primaryantibody. For example, a system of sandwich EIA can be constructed bypreparing two kinds of antibodies which are specific for the marker andare different in the epitope, immobilizing one of them on a carrier as aprimary antibody, and labeling the other as a secondary antibody with anenzyme. In addition, a system of an immunoassay by a binding inhibitionmethod or a competition method can also be constructed. Further, when asubstrate is used as a carrier, an immunoassay by an antibody chip ispossible. With the antibody chip, the concentrations of a plurality ofmarkers can be measured simultaneously, and rapid measurement ispossible.

On the other hand, in one embodiment, when mass spectrometry is used ina method for measuring a marker, the marker can be trapped on a carrierby an ionic bond or hydrophobic interaction, in addition to an antibody.An ionic bond or hydrophobic interaction has no higher specificity thanthat of bioaffinity such as that between an antigen and antibody, and asubstance other than the marker is trapped, but they have no problemsince mass spectrometry performs quantitation by a mass spectrometerspectrum reflecting the molecular weight. Particularly, whensurface-enhanced laser desorption/ionization-time-of-flight massspectrometry (referred to as “SELDI-TOF-MS” as used herein) is performedusing a protein chip employing a substrate as a carrier, theconcentration of the marker can be measured more precisely. As the kindof substrate which can be used, a cation exchange substrate, an anionexchange substrate, a normal phase substrate, a reverse phase substrate,a metal ion substrate, an antibody substrate and the like can be used,and a cation exchange substrate, particularly, a weak cation exchangesubstrate and a metal ion substrate are preferably used.

When the marker is trapped on a carrier by an ionic bond, an ionexchanger is immobilized on a carrier. In this case, as an ionexchanger, either of an anion exchanger and a cation exchanger can beused, and further, any of a strong anion exchanger, a weak anionexchanger, a strong cation exchanger, and a weak cation exchanger can beused. Examples of the weak anion exchanger include exchangers having aweak anion exchange group such as dimethylaminoethyl (DE) anddiethylaminoethyl (DEAE). In addition, examples of the strong anionexchanger include exchangers having a strong anion exchange group suchas quaternary ammonium (trimethylaminomethyl) (QA), quaternaryaminoethyl (diethyl, mono-2-hydroxybutylaminoethyl) (QAE), andquaternary ammonium (trimethylammonium) (QMA). In addition, examples ofthe weak cation exchanger include exchangers having a weak cationexchange group such as carboxymethyl (CM). Further, examples of thestrong cation exchanger include exchangers having a strong cationexchange group such as sulfopropyl (SP). On the other hand, when themarker is trapped on a carrier by hydrophobic interaction, a substancehaving a hydrophobic group is immobilized on a carrier. Examples of thehydrophobic group include a C4-C20 alkyl group and a phenyl group.Further, the marker can be trapped on a carrier on which a metal ionsuch as Cu²⁺, Zn²⁺, Ni²⁺, Ca²⁺, Co²⁺, or Mg²⁺ is immobilized.

In one embodiment, as an example of a carrier to be used, known carrierssuch as beads, a microtiter plate, and a resin can be employed.Particularly, beads and a microtiter plate have previously been used inan immunoassay, and construction of a measuring system is easy. On theother hand, a carrier having a flat part such as a substrate can also beused. In this case, it is preferable to immobilize a substance havingaffinity for the marker on a part of the flat part. As an example, therecan be mentioned a carrier including a chip as a substrate, and anantibody specific for the marker is immobilized on a plurality of placeson the chip in a spot-like manner.

Since a cell which was selected by using, as an index, the detectionagent or the diagnostic agent such as a probe, a primer, or an antibodyin accordance with the present invention is an undifferentiated cell ora precursor cell having a proliferation ability (e.g., anundifferentiated cell or a precursor cell existing in a cornealendothelial cell), it is preferable for treating or preventing a cornealendothelial disease, disorder or condition such as bullous keratopathyor corneal endotheliitis or other ophthalmological diseases, from theviewpoint of safety, the survival rate, and the network forming ability,as compared with the previous miscellaneous cell populations or aprecursor cell in which a foreign gene is introduced. Since a cell whichcan be selected is an undifferentiated cell or a precursor cell beforedivision arrest, that is, during proliferation, and has a possibilitythat it is differentiated and matured at an optimal place in a brain,and there is a possibility that an undifferentiated cell or a precursorcell further proliferates in vivo, a therapeutic effect for a longerterm can be expected. Therefore, it is possible to state that thepresent invention paves the way for practical application of effectivetreatment or prevention of a corneal endothelial disease, disorder orcondition such as bullous keratopathy or corneal endotheliitis or otherophthalmological diseases.

(Screening)

The detection method in accordance with the present invention can beapplied to screening of a substance effective in inducingdifferentiation into a cell having a high proliferation ability and/oran undifferentiated cell existing in corneal endothelial cells. That is,by determining whether or not a cell was differentiation-induced into acell having a high proliferation ability and/or an undifferentiated cellexisting in corneal endothelial cells by addition of a test substanceusing, as an index, expression of GPR49/LGR5, and/or the factors of theHedgehog pathway such as SHH, PTCH1, GLI1, and GLI2, and/or the factorsof the Wnt pathway such as LRP6 and β-catenin, a substance effective ininducing differentiation into a cell having a high proliferation abilityand/or an undifferentiated cell existing in corneal endothelial cellscan be screened.

Therefore, according to the present invention, a method for screening asubstance effective in inducing differentiation into a cell having ahigh proliferation ability and/or an undifferentiated cell existing incorneal endothelial cells is provided, and this method includes (i) astep of contacting a test substance with a cell (e.g, an ES cell or aniPS cell) which can differentiate into a cell having a highproliferation ability and/or an undifferentiated cell existing incorneal endothelial cells; and (ii) a step of detecting expression ofGPR49/LGR5, and/or the factors of the Hedgehog pathway such as SHH,PTCH1, GLI1, and GLI2, and/or the factors of the Wnt pathway such asLRP6 and β-catenin in the cell after contact with the test substance. Inthe step (i), the cell which can differentiate into a cell having a highproliferation ability and/or an undifferentiated cell existing incorneal endothelial cells can be preferably collected from a culturedcell containing an iPS cell and an ES cell, or a neural precursor cellor a neural crest cell which was differentiation-induced from thesecells.

In the step (i), “contacting a test substance” can be performed byadding a test substance to a cultured cell containing a cell which candifferentiate into a cell having a high proliferation ability and/or anundifferentiated cell existing in corneal endothelial cells.

Examples of the usable “test substance” include, but are not limited toa synthetic low-molecular weight compound, a protein, a syntheticpeptide, a purified or partially purified polypeptide, an antibody, abacterium-releasing substance (including a bacterial metabolite), and anucleic acid (antisense, ribozyme, RNAi, etc.), preferably, a compoundor a salt thereof or a solvate thereof (e.g., a hydrate). The “testsubstance” may be a novel substance, or may be a known substance.

In the step (ii), according to the detection method of the presentinvention, expression of GPR49/LGR5, and/or the factors of the Hedgehogpathway such as SHH, PTCH1, GLI1, and GLI2, and/or the factors of theWnt pathway such as LRP6 and β-catenin can be detected.

As a specific embodiment, by appropriately implementing detectionutilizing a hybridization method, detection utilizing a nucleic acidamplification method, and detection utilizing an antigen-antibodyreaction described herein, expression of GPR49/LGR5, and/or the factorsof the Hedgehog pathway such as SHH, PTCH1, GLI1, and GLI2, and/or thefactors of the Wnt pathway such as LRP6 and β-catenin can be detected.

In the step (ii), when the test substance is contacted with the cell,and expression of GPR49/LGR5, and/or the factors of the Hedgehog pathwaysuch as SHH, PTCH1, GLI1, and GLI2 is detected in a cell sample, it canbe determined that the substance is a substance effective in inducingdifferentiation into a cell having a high proliferation ability and/oran undifferentiated cell existing in corneal endothelial cells. Whensuppression or disappearance of expression of the factors of the Wntpathway such as LRP6 and β-catenin is detected, it can be determinedthat the substance is a substance effective in inducing differentiationinto a cell having a high proliferation ability and/or anundifferentiated cell existing in corneal endothelial cells.

A substance specified by the screening method in accordance with thepresent invention can be used as a substance effective in inducingdifferentiation into a cell having a high proliferation ability and/oran undifferentiated cell existing in corneal endothelial cells.According to the present invention, a method for screening a substanceeffective in inducing differentiation into a cell having a highproliferation ability and/or an undifferentiated cell existing incorneal endothelial cells is provided, and this method may furthercomprise (iii) a step of detecting expression of a differentiationmarker gene other than GPR49/LGR5, and/or the factors of the Hedgehogpathway such as SHH, PTCH1, GLI1, and GLI2, and/or the factors of theWnt pathway such as LRP6 and β-catenin, in the cell after contact withthe test substance. When expression of GPR49/LGR5, and/or the factors ofthe Hedgehog pathway such as SHH, PTCH1, GLI1, and GLI2, and/or thefactors of the Wnt pathway such as LRP6 and β-catenin is detected in thestep (ii), and expression of a differentiation marker gene (e.g., Ki-67or BrdU) other than GPR49/LGR5, and/or the factors of the Hedgehogpathway such as SHH, PTCH1, GLI1, and GLI2, and/or the factors of theWnt pathway such as LRP6 and β-catenin is detected in the step (iii),the substance can be precisely determined to be a substance effective ininduction into a cell having a high proliferation ability and/or anundifferentiated cell existing in corneal endothelial cells. Inaddition, the step (iii) has only to be performed after the step (i),and may be performed before or after the step (ii).

(General Technique)

A molecular biological procedure, a biochemical procedure and amicrobiological procedure as used herein are well-known andconventionally used in the art, and are described, for example, inSambrook J. et al. (1989). Molecular Cloning: A Laboratory Manual, ColdSpring Harbor and its 3rd Ed. (2001); Ausubel, F. M. (1987). CurrentProtocols in Molecular Biology, Greene Pub. Associates andWiley-Interscience; Ausubel, F. M. (1989). Short Protocols in MolecularBiology: A Compendium of Methods from Current Protocols in MolecularBiology, Greene Pub. Associates and Wiley-Interscience; Innis, M. A.(1990). PCR Protocols: A Guide to Methods and Applications, AcademicPress; Ausubel, F. M. (1992). Short Protocols in Molecular Biology: ACompendium of Methods from Current Protocols in Molecular Biology,Greene Pub. Associates; Ausubel, F. M. (1995). Short Protocols inMolecular Biology: A Compendium of Methods from Current Protocols inMolecular Biology, Greene Pub. Associates; Innis, M. A. et al. (1995).PCR Strategies, Academic Press; Ausubel, F. M. (1999). Short Protocolsin Molecular Biology: A Compendium of Methods from Current Protocols inMolecular Biology, Wiley, and annual updates; Sninsky, J. J. et al.(1999). PCR Applications: Protocols for Functional Genomics, AcademicPress, and Experimental Medicine, separate volume, “Gene Introduction &Expression Analysis Experimental Method” Yodosha Co., Ltd., 1997, therelevant part (which can be the whole) of them is incorporated herein byreference.

A DNA synthesis technique for producing an artificially synthesized geneand nucleic acid chemistry are described, for example, in Gait, M. J.(1985). Oligonucleotide Synthesis: A Practical Approach, IRL Press;Gait, M. J. (1990). Oligonucleotide Synthesis: A Practical Approach, IRLPress; Eckstein, F. (1991). Oligonucleotides and Analogues: A PracticalApproach, IRL Press; Adams, R. L. et al. (1992). The Biochemistry of theNucleic Acids, Chapman & Hall; Shabarova, Z. et al. (1994). AdvancedOrganic Chemistry of Nucleic Acids, Weinheim; Blackburn, G. M. et al.(1996). Nucleic Acids in Chemistry and Biology, Oxford University Press;and Hermanson, G. T. (1996). Bioconjugate Techniques, Academic Press,the relevant part of them is incorporated herein by reference.

For example, as used herein, the oligonucleotide of the presentinvention can also be synthesized using, for example, an automated DNAsynthesizer (e.g., a synthesizer commercially available from Biosearch,Applied Biosystems) by a standard method known in the art. For example,a phosphorothioate-oligonucleotide can also be synthesized by the methodof Stein et al. (Stein et al., 1988, Nucl. Acids Res. 16: 3209), and amethyl phosphonate-oligonucleotide can also be prepared by using apore-adjusted glass polymer support (Sarin et al., 1988, Proc. Natl.Acad. Sci. USA 85: 7448-7451).

Reference literatures such as scientific literatures, patents, andpatent applications cited herein are incorporated herein by reference tothe same extent that the entirety of them is specifically described.

As described above, the present invention has been illustrated byshowing preferable embodiments for ease of understanding. The presentinvention will be illustrated below based on Examples, but theaforementioned illustration and the following Examples are provided onlyfor the purpose of exemplification, and are not provided for the purposeof limiting the present invention. Therefore, the scope of the presentinvention is not limited to embodiments and Examples specificallydescribed herein, and is limited only by the scope of claims.

EXAMPLES

If necessary, handling of animals used in the following Examples wereperformed in compliance with a standard defined in Kyoto PrefecturalUniversity of Medicine or Doshisha University, and based on HelsinkiDeclaration. In addition, according to the ARVO Statement for the Use ofAnimals in Ophthalmic and Vision Research, animals were fed and handled.In addition, as reagents, products specifically described in Exampleswere used, but equivalent products of other manufacturers (Sigma, WakoPure Chemical Industries Co., Ltd., Nacalai Tesque, Inc., abcam, SantaCruz Biotechnology, R & D Systems, Abnova, Assay Pro, Origene, Biobyt,Biorad, Cell Signaling Technology, GE Healthcare, IBL, and the like) canbe used as substitutes.

(Experimental Material and Method) (Material) (Corneal Tissue)

All human corneal tissues used in the present experiment were tissuesimported from SightLife™ (Northwest Lions Foundation) of AmericanSeattle Eye Bank. All monkey corneal tissues used were tissues of acynomolgus monkey euthanized for other research purposes (Nissei BilisCo., Ltd., Ohtsu, Japan, or Keari Co., Ltd., Wakayama, Japan). Allcorneas were preserved at 4° C. in a preservation medium (Optisol;Chiron Vision Corporation, Irvine, Calif.). All experiments wereperformed according to doctrine of Helsinki Declaration.

(Cell Culture)

In primary culture of a human corneal endothelial cell, a Descemet'smembrane containing an endothelial cell layer was peeled from a cornealtissue, and placed into 2 mg/ml Collagenase A (catalog No.: 70164923;Roche Applied Science, Penzberg, Germany) dissolved in OPTIMEM-I, andincubated at 37° C. After 3 hours, the resultant was centrifuged at 1000rpm for 5 minutes to remove the supernatant, a culture medium was addedto a precipitated corneal endothelial cell mass for admixing, and thetotal amount was seeded on a 12-well plate coated with FNC Coating Mix(catalog No.: 0407; Athena Enzyme Systems, Baltimore, Md., USA). As aculture medium, OPTIMEM-I (catalog No.: 51985; Gibco-Invitrogen,Carlsbad, Calif.), to which 5% fetal bovine serum (catalog No.:10437-028; fetal bovine serum; FBS; BioWest, France), 50 μg/mlGentamicin (Invitrogen), and 10 μg/ml Y-27632 (Calbiochem, La Jolla,Calif.) were added, was used.

In primary culture of a monkey corneal endothelial cell, a Descemet'smembrane containing an endothelial cell layer was peeled from a cornealtissue, placed in 1.2 U/ml Dispase I [(Sanko Pure Chemical Co., Ltd.)catalog No.: GD81060] dissolved in DMEM (Gibco-Invitrogen), andincubated at 37° C. After 1 hour, a corneal endothelial cell was peeledand recovered from a Descemet's membrane by pipetting, and the resultantwas centrifuged at 1000 rpm for 5 minutes to remove the supernatant. Aculture medium was added to a precipitated corneal endothelial cell foradmixing, and the total amount was seeded on a 6-well plate coated withFNC Coating Mix. As a culture medium, DMEM (catalog No.: 12320;Gibco-Invitrogen), to which 10% FBS, 50 μg/ml Gentamicin (catalog No.:15710-064; Invitrogen), 10 μg/ml Y-27632 (catalog No.: 6880005;Calbiochem, La Jolla, Calif.), and a 2 ng/ml basic fibroblast growthfactor (catalog No.: 13256-029; bFGF; Invitrogen) were added, was used.

As a human cornea, a cornea, in which the period before primary culturewas less than 14 days, was used. For culture of human and monkey cornealendothelial cells (CEC), a system previously reported [Tan D T et al.,Lancet., 2012; 379: 1749-1761; Koizumi N et al., Exp Eye Res., 2012; 95:60-67; Koizumi N et al., Invest Ophthalmol Vis Sci. 2007; 48: 4519-4526;Okumura N Et al., Am J Pathol. 2012; 181: 268-277] was used.

A medium was exchanged every 2 days. Subculture was performed at thetime point of 50 to 80% confluent. As a subculture method, cells werewashed with Ca²⁺Mg²⁺-not containing (free) PBS (PBS-; NissuiPharmaceutical Co., Ltd., Tokyo, Japan), TrypLE™ Select (catalog No.:12563; Invitrogen) was added, and the resultant was incubated at 37° C.for 5 minutes. Cells were peeled and recovered from the plate, andcentrifuged at 1000 rpm for 5 minutes, and a culture medium was added toprovide a cell suspension. Cells were seeded on a plate coated with FNCCoating Mix at a density of 1:2.

(Immunostain)

Immunohistochemical study was performed according to the methodpreviously described by the present inventors [Nakamura T et al., StemCells, 2007; 25: 628-638; Nakamura T et al., Stem Cells, 2008; 26:1265-1274].

A corneal tissue was embedded with OCT compound (catalog No.: 4583;Sakura Finetek, Tokyo, Japan), and frozen in liquid nitrogen to preparea frozen corneal tissue section. The frozen block was sliced to 8 μm,applied on a silane-coating slide, and air-dried. For preparing thewhole tissue section, a Descemet's membrane containing a cornealendothelial cell was peeled from a corneal tissue, allowed to stand inice-cooled 100% acetone for 30 seconds, applied on a silane-coatingslide, and air-dried. As a cultured corneal endothelial cell, a cellcultured on a LabTek 8-well plastic chamber slide was used. Ice-cooled100% acetone was added to a corneal tissue and the cultured cell, theresultant was allowed to stand at 4° C. for 15 minutes to fix the cell.After shaking and washing with 0.15% TritonX/PBS-, 1% bovine serumalbumin (catalog No.: A4503BSA; Sigma-Aldrich, St. Louis, Mo.) wasadded, and allowed to stand at room temperature for 30 minutes toperform blocking. A primary antibody was diluted with 1% BSA, andincubated at room temperature for 1 hour. As a primary antibody to beused, anti-rabbit GPR49/LGR5 (catalog No.: GTX71143; 1: 200; GeneTexInc., San Antonio, Tex.), anti-rabbit Nestin (catalog No.: PRB-570C; 1:200; COVANCE, Berkeley, Calif.), anti-mouse ABCG2 (catalog No.: NC 236;1: 2; Kamiya biomedical CO., Seattle, Wash. USA), anti-mouse Ki-67(catalog No.: 556003; 1: 200 BD Pharmingen™ NJ, USA), anti-rabbit Na⁺/K⁺ATPase (catalog No.: A132; 1: 100; Zymed), and anti-rabbit ZO1 (ZymedLaboratries Inc., South San Francisco, Calif.) were used. The resultantwas washed with 0.15% Triton/PBS- twice, and PBS- once.

A secondary antibody was diluted with a mixed solution of 1% BSA and0.15% TrironX/PBS-, and incubated at room temperature for 30 minutes. Asa secondary antibody to be used, Alexa™ Fluor 488-labeled (conjugated)goat anti-rabbit IgG (catalog No.: A11034; 1: 1500; MolecularProbe-Invitrogen) and Alexa™ Fluor 488-labeled (conjugated) goatanti-mouse IgG (catalog No.: A11029; 1: 1500; MolecularProbe-Invitrogen) were used. The secondary antibody was shaken andwashed with 0.15% Triton/PBS- twice, and PBS- once, subjected to nuclearstaining with propidium iodide (catalog No.: SP29004-41; PI; NacalaiTesque, Inc. Kyoto, Japan), and embedded while covered with a coverglass. This was observed with a confocal laser microscope (OlympusFluoview, Tokyo, Japan), and a photograph was taken.

(Real Time PCR)

A real time PCR experiment was performed according to the methodpreviously described by the present inventors [Nakamura T et al., StemCells., 2007; 25: 628-638].

For extracting RNA from a corneal tissue and a cultured cell, RNeasyMini Kit (catalog No.: 74106; QIAGEN, Valencia, Calif.) was used. Addedwas 1 μl of OligodT Primer (catalog No.: 18418-020; Invitrogen) per 1 μgof the extracted RNA, and the resultant was incubated at 70° C. for 2minutes, and immediately ice-cooled. Further, a reaction liquidincluding 5× First-Strand buffer, 100 mM DTT (catalog No.: 772590;Invitrogen), SuperScript™ II Reverse Transcriptase (catalog No.:18064-014; Invitrogen), 2.5 mM dNTP (catalog No.: BG7401A; Takara BioInc., Otsu, Japan), and 40 unit/μl RNase Inhibitor (catalog No.:SIN-101; Toyobo Co., Ltd., Osaka Japan) was added, and incubated at 42°C. for 45 minutes and 70° C. for 15 minutes to synthesize cDNA. Forpreparing a primer, BLAST programs (NCBI) were utilized (Table 1). Aprimer and SYBR (registered trademark) Green PCR Master Mix (catalogNo.: 4367659; Applied Biosystems, Foster City, Calif.) were added to 2μl of the synthesized cDNA, and the resultant was reacted on ABI Prism7000 (Applied Biosystems) at 40 cycles of 95° C. for 15 seconds and 60°C. for 1 minute. The resulting data were analyzed by ABI PRISM(registered trademark) 7000 Sequence Detection System. In addition, theamount of expression of each gene was corrected with the amount of agene expressing β-actin, and was shown as a relative value to a control.

TABLE 1 GenBank Accession Gene Number Sequence Human GPR49 NM_003667Forward 5′-GAGGATCTGGTGAGCCTGAGAA-3′ (SEQ ID NO: 13) Reverse5′-CATAAGTGATGCTGGAGCTGGTAA (SEQ ID NO: 14) Human Nestin NM_006617Forward 5′-ATGCTCCTCTCTCTCTGCTCCA (SEQ ID NO: 15) Reverse5′-CTAGTGTCTCATGGCTCTGGTTTTTC (SEQ ID NO: 16) Human ABCG2 NM_04827.2Forward 5′-AGTGGCTTTCTACCTTGTCG (SEQ ID NO: 17) Reverse5′-ACAGAAACCACACTCTGACC (SEQ ID NO: 18) Human SHH NM_000193.2 Forward5′ACGGCCCAGGGCACCATTCT (SEQ ID NO: 19) Reverse 5′-GGACTTGACCGCCATGCCCA(SEQ ID NO: 20) Human Ptch1 NM_000264.3 Forward5′TCGCTCTGGAGCAGATTTCCAAGGG (SEQ ID NO: 21) Reverse5′-GCAGTCTGGATCGGCCGGATTG (SEQ ID NO: 22) Human Smo NM_005831.4 Forward5′-GTGAGTGGCATTTGTTTTGTGGGC (SEQ ID NO: 23) Reverse5′-CAGGCATTTCTGCCGGGGCA (SEQ ID NO: 24) Human Gli1 NM_005269.2 Forward5′-GCCCCCATTGCCCACTTGCT (SEQ ID NO: 25) Reverse 5′-TGCAGGGGACTGCAGCTCC(SEQ ID NO: 26) Human Gli2 NM_005270.4 Forward 5′-GGCCGCCTAGCATCAGCGAG(SEQ ID NO: 27) Reverse 5′-CACCGCCAGGTTGCCCTGAG (SEQ ID NO: 28)Human beta- NM_001101 Forward 5′-GGACTTCGAGCAAGAGATGG (SEQ ID NO: 29)actin Reverse 5′-ATCGCTGGAAGGTGGACAG (SEQ ID NO: 30)

(Flow Cytometry)

A cultured monkey corneal endothelial cell was seeded on a 6-well platecoated with FNC Coating Mix, and cultured under conditions of 37° C. and5% CO₂ for 14 days. Cells were peeled with TrypLE™ Select and recovered.For fractionating GPR49/LGR5-positive cells, 1% BSA was added torecovered cells, and incubated at room temperature for 15 minutes toperform blocking. As a primary antibody, anti-rabbit GPR49/LGR5 andAlexa™ Fluor 488 labeled goat anti-rabbit IgG <concerning both of them,see the above Example> were used, and each was incubated at roomtemperature for 20 minutes. Using a flow cytometer (FACS Aria II (BDBiosciences, Franklin Lakes, N.J.)), GPR49/LGR5-positive cells andGPR49/LCR5-negative cells were fractionated, and the respective cellswere seeded on a 8-well chamber slide, and cultured. After 3 days, thecells were immunostained with anti-mouse Ki-67, and the number ofKi-67-positive cells was counted.

For studying the cell proliferation rate in GPR49/LGR5-positive cells,70% ethanol was added to recovered cells, and the resultant wasincubated at −20° C. for 2 hours to fix the cells. After washing withPBS- twice, 1% BSA was added, and incubated at room temperature for 15minutes to perform blocking. As a primary antibody, anti-rabbitGPR49/LGR5 (1:100) and anti-mouse Ki-67 (1:100) were used, and theresultant was incubated at room temperature for 30 minutes. As asecondary antibody, Alexa™ Fluor 488-conjugated goat anti-rabbit IgG(1:1500) and Alexa™ Fluor 594-conjugated goat anti-mouse IgG (1:1500;Molecular Probe-Invitrogen) were used, and the resultant was incubatedat room temperature for 20 minutes. For sorting cells, FACS Aria II (BDBio-sciences, Franklin Lakes, N.J.) was used. For analysis of data, anattached software was used.

(Measurement of Cell Area)

Each isolated cell fraction was centrifuged, and re-suspended in aculture medium. Cells (about 100 cells/ml) were placed in a 6-wellplate, and photographed under an inverted microscope. The areas of cellswere randomly measured using the Scion Image software (200cells/fraction), and statistically analyzed [Nakamura T et al., StemCells., 2007; 25: 628-638].

(Gene Introduction; RNA Interference)

For preparing a lentivirus particle containing GPR49/LGR5 shRNA virusvector, GPR49/LGR5 MISSION (registered trademark) shRNA Plasmid DNA(Sigma-Aldrich) was purchased (Table 2).

TABLE 2 shRNA Clone IDInsert sequence (Mspi-Sense-loop-antisense-terminator shGPR49-587NM_003667.2-CCGGCCGTCTGCAATCAGTTACCTACTCGAGTAGGTAACTGATTGCAGACGGTTTTT (SEQ ID NO: 31)1052s1c1 shGPR49-588 NM_003667.2-CCGGCTTACATTTATCAGTCCTGAACTCGAGTTCAGGACTGATAAATGTAAGTTTTT (SEQ ID NO: 32)2425s1c1 shGPR49-589 NM_003667.2-CCGGGCTCTACTGCAATTTGGACAACTCGAGTTGTCCAAATTGCAGTAGAGCTTTTT (SEQ ID NO: 33)2286s1c1 Non-Target noneCCGGCAACAAGATGAAGAGCACCAACTCGAGTTGGTGCTCTTCATCTTGTTGTTTTT (SEQ ID NO: 34)(NT)

In addition, as a GPR49/LGR5 expression vector, a vector assigned fromSatoshi Kawasaki, Ophthalmology Department, Kyoto Prefectural Universityof Medicine was used. A method for preparing this vector is as follows.

That is, for constructing this vector, a lentivirus plasmid vectorexpressing an objective gene (herein, GPR49/LGR5) was used. As a vectorfor inserting the objective gene, the present inventors used acommercially available lentivirus vector (pLenti6.3_V5-TOPO;Invitrogen). An amplification reaction was performed using a primer pairincluding the whole coding sequence of a specified gene and using cDNAas a template, and the resultant was gel-purified, and ligated with alentivirus plasmid vector.

Upon expression of this lentivirus, for production and infection, aprotocol used for shRNA previously reported by a part of the presentinventors (Nakatsukasa M. Kawasaki S, Yamasaki K, Fukuoka H, Matsuda A,Tsujikawa M, Tanioka H, Nagata-Takaoka M, Hamuro J, Kinoshita S, Am JPathol. 2010 September; 177 (3): 1344-55.) was modified for use. Inbrief, lentivirus plasmid DNA was transfected into HEK 293T cells usingViraPower™ Lentiviral Packaging Mix (Invitrogen) which was a packagingplasmid mixture containing pLP1, pLP2 and pLP/VSVG plasmids, and 14 μlof Fugene HD as a transfection reagent. After 18 hours, a medium wasremoved by suction, and replaced with a complete medium (DMEMsupplemented with 10% FBS; hereinafter, also referred to as “culturemedium”), and the quality of lentivirus particles was evaluated.

More particularly, HEK293T cells were seeded on a culture mediumobtained by adding 10% FBS to DMEM, at a density of 9.0×10⁵ cells/25cm². After culture for 24 hours, OPTIMEM-I, FuGENE (registeredtrademark) HD Transfection Reagent (Roche Applied Science) andLentiviral Packaging Mix (Sigma-Aldrich (MISSION Lentiviral PackagingMix) or Invitrogen) were added, and the resultant was incubated underconditions of 37° C. and 5% CO₂. After 18 hours, the medium wasexchanged with a culture medium, and cells were cultured underconditions of 37° C. and 5% CO₂. After 24 hours, a culture mediumcontaining lentivirus particles was recovered. For measuring the titerof the recovered lentivirus, the HIV-1 p24 Antigen ELISA kit(ZeptoMetrixCorporation, Buffalo, N.Y., USA) was used. In addition, as acontrol, Non-Target shRNA Control Vector (Sigma-Aldrich) having a randomsequence was used. Cultured human and monkey corneal endothelial cellswere seeded on a 6-well plate (5000 cells/well; 6-well plate coated withFNC Coating Mix (registered trademark)) or an 8-well chamber slide (500cells/well), and cultured under conditions of 37° C. and 5% CO₂. After24 hours, lentivirus particles prepared and 4 μg/ml hexadimethrinebromide (Sigma-Aldrich, also referred to as Polybrene) were added, andthe cells were transfected under a conditions of 37° C. and 5% CO₂.After 24 hours, a medium was exchanged with a culture medium containing0.4 μg/ml puromycin (Calbiochem), and a puromycin-resistant cell wasselected. A puromycin-resistant colony was cultured in the presence of0.4 μg/ml puromycin, and a medium was exchanged every 2 days.

(Construction of Lentivirus Plasmid Vector for Gene Expression)

For constructing a lentivirus plasmid vector expressing an objectivegene, a commercially available lentivirus vector (pLenti6.3_V5-TOPO;Invitrogen) was used. A primer pair surrounding the whole codingsequence of a specified gene was used to amplify cDNA, gel-purified and,then, ligated into a lentivirus plasmid vector.

Production and infection of a lentivirus for expression were performedby a modified version of a protocol by the present inventors, which wasused with respect to shRNA [Nakatsukasa M et al., Am J Pathol., 2010;177: 1344-1355]. In brief, lentivirus plasmid DNA was transfected,together with a plasmid packaging plasmid mixture ViraPower™ LentiviralPackaging Mix (Invitrogen) containing pLP1, pLP2 and pLP/VSVG plasmids,into HEK293T cells by using FuGENE (registered trademark) HD as atransfection reagent. After 18 hours, the medium was sucked andexchanged with a complete medium, and the amount of lentivirus particleswas evaluated.

(Gene Introduction)

The culture supernatant containing virus particles having infectionability were recovered, and transferred to human CEC of 5000 cells/wellin a 6-well plate containing FNC Coating Mix (registered trademark). Thesupernatant was applied on cultured CEC in the presence of 4 μg/mlPolybrene. Upon collection of a puromycin-resistant colony, cells werecultured in the presence of 0.4 μg/ml puromycin, and the medium wasexchanged every 2 days.

(Western Blotting)

For Western blotting, the following rabbit polyclonal antibodies:anti-LRP6, p-LRP6 (Cell Signaling Technology, Inc., Beverly, Mass.), aswell as the following mouse monoclonal antibodies: β-catenin (BDBiosciences) and β-actin (Sigma-Aldrich, St. Louis, Mo.); were used. Asa secondary antibody, HRP-labeled anti-rabbit or mouse IgG (GEHealthcare, Piscataway, N.J.) was used. Recombinant human SHH,purmorphamine, cyclopamine and RSpos were purchased from R&D SystemsInc. (Minneapolis, Minn.).

Cultured human CEC was washed with PBS, and then, rinsed with a lysisbuffer containing PBS, 1% TRITON™ X-100, 0.5 M EDTA, phosphataseinhibitor cocktail 2 (Sigma-Aldrich) and phosphatase inhibitor cocktail(Roche). Detection of activated β-catenin (non-membrane-bound type) wasimplemented according to the protocol previously reported [Aghib D F etal., Exp Cell Res., 1995; 218: 359-369]. In brief, a cell lysate treatedwith Con A Sepharose™ 4B (GE Healthcare) was incubated at 4° C. for 1hour. After centrifugation at 4° C. for 10 minutes, the supernatant wastransferred into a new tube, Con A Sepharose™ was added to each tube,and the resultant was incubated at 4° C. for 1 hour. Finally, aftersimple centrifugation, the supernatant was transferred into a new tube,and the protein concentration was determined.

Then, the protein was separated by SDS-PAGE, and transferred to a PVDFmembrane. Thereafter, the membrane was blocked with 1% ECL AdvanceBlocking Reagent (GE Healthcare) in a TBS-T buffer, and incubated at 4°C. overnight together with a primary antibody. After washing in a TBS-Tbuffer three times, the PVDF membrane was incubated together with anappropriate HRP-labeled anti-rabbit or mouse IgG secondary antibody atroom temperature for 1 hour. The membrane was photosensitized with ECLAdvance Western Blotting Detection Kit (GE Healthcare), and observed byLAS3000S imaging system. (Fuji Film Co., Ltd., Tokyo).

Each Example and results are shown below.

Example 1 Expression of Stem Cell Marker in Whole Layer Human CornealTissue

An in vivo expression pattern of GPR49/LGR5 in human CEC wasinvestigated by an indirect immunostaining method. For comparison,Nestin and ATP binding set subfamily G member 2 (ABCG2) which weremarkers of an immature cell and a precursor cell were also investigated.

In order to retrieve a protein to be expressed specifically for cornealendothelial cells, immunostaining was comprehensively performed usingthe already reported stem cell marker (FIG. 2a ). As a result, strongexpression of GPR49/LGR5 was recognized specifically for cornealendothelial cells. As a comparison subject, Nestin (Lendahl U et al.,Cell, 1990) which was an undifferentiated cell marker and ABCG2 (Chen etal., Stem Cells, 2004) which was expressed specifically for limbuscorneae basal cells were used.

When CEC of these tissues were investigated in this manner, strongexpression of GPR49/LGR5 was observed, particularly, in a peripheralregion. However, GPR49/LGR5 was only minimally expressed in a cornealepithelium and a corneal stroma (FIG. 2a ). It was found that Nestin isexpressed over the whole cornea to a moderate degree, and that ABCG2 isexpressed mainly in the basal cell layer of a corneal epithelium, and isweakly expressed in a stroma and an endothelium of a cornea (FIG. 2a ).

Strong expression of Nestin was observed in the whole corneal layer.Strong expression of ABCG2 was observed in corneal epithelial basalcells, and expression was also observed in a corneal parenchyma and acorneal endothelium. The amounts of expression of mRNA of GPR49/LGR5,Nestin and ABCG2 were compared by real time PCR (FIG. 2b ). The amountsof expression of Nestin mRNA and ABCG2 mRNA were significantly elevatedin a corneal epithelium and a corneal parenchyma as compared with acorneal endothelium. On the other hand, the amount of expression ofGPR49/LGR5 mRNA was significantly elevated in a corneal endothelium.

In this way, real time PCR showed that the average GPR49/LGR5 mRNAexpression was significantly upregulated in CEC as compared withparenchymal cells and epithelial cells of a cornea (*p<0.05) (FIG. 2b ).To the contrary, the expression levels of Nestin and ABCG2 in theparenchymal cell and the epithelial cell of a cornea were higher thanthe expression levels in CEC (FIG. 2b ). Therefore, it was found thatexpression of GPR49/LGR5 is most remarkable in CEC among cornealtissues.

Therefore, GPR49/LGR5 was determined to be one of proteins which wereexpressed specifically for corneal endothelial cells.

Example 2 Localization of Expression of GPR49/LGR5 in Human CornealWhole Tissue Section

Then, the present inventors studied a localization pattern of GPR49/LGR5using the whole mount immunofluorescence method.

In the present Example, in order to investigate localization ofexpression of GPR49/LGR5 in a corneal endothelial tissue, a Descemet'smembrane containing corneal endothelial cells was peeled from a cornealtissue, and the amount of expression was compared and studied byimmunostaining and real time PCR (FIG. 2c ).

As a result of the immunostaining, as shown in FIGS. 2d to e , strongexpression of GPR49/LGR5 was recognized in a cell membrane and acytoplasm of an endothelial cell in a corneal periphery (FIG. 2d ). Inaddition, the amount of expression of GPR49/LGR5 mRNA was also elevatedin a peripheral corneal endothelium as compared with the central portion(FIG. 2e ).

It was found that expression of GPR49/LGR5 is increased in a peripheralregion, in CEC, and that the expression level thereof is graduallydecreased towards the central region of CEC (FIGS. 2c, d ). Real timePCR clearly showed that expression of GPR49/LGR5 in a peripheral regionis upregulated as compared with the central region (diameter 7 mm)(*p<0.05) (FIG. 2e ). These findings show that GPR49/LGR5 is inherentlyexpressed in peripheral CEC, in a corneal tissue.

Example 3 Expression of GPR49/LGR5 in Cultured Human Corneal EndothelialCell

In the present Example, expression of GPR49/LGR5 in a cultured humancorneal endothelial cell was investigated.

A human corneal endothelial cell is poor in the proliferation ability ina living body, and cell culture outside a living body is also extremelydifficult. Previously, a variety of culture methods have been studied,but a subculture method in the state where a hexagonal cobblestone cellform is maintained has not been established. Then, the amount ofexpression of GPR49/LGR5 upon culture of a human corneal endothelialcell was studied by the existing method. As a comparison subject, Nestinwas used.

As a result of immunostain, in a corneal tissue (in vivo), very strongexpression was observed in both of GPR49/LGR5 and Nestin (FIG. 3a ). Ina primary cultured cell, expression of GPR49/LGR5 was not recognized,but expression of Nestin was confirmed. In a subculture cell (in vitroP0), expression of GPR49/LGR5 could not be confirmed, but expression ofNestin was confirmed, although expression was reduced. In addition, in asubculture cell (in vitro P1), cell differentiation was made, and anincrease in nucleous was observed. When the amount of each mRNA of acorneal endothelial cell cultured under the same condition was analyzed,the amount of expression of GPR49/LGR5 mRNA was significantly decreasedin the culture envelopment, but little decrease in the amount ofexpression of Nestin mRNA was recognized (FIG. 3b ).

In this way, a phase contrast microphotograph of in vivo human CECrevealed that these presented a confluent monolayer of uniform hexagonalcells having a smaller size (FIG. 3a ). To the contrary, it was foundthat cultured CEC (P0, P1) is extended, and is not uniform hexagon (FIG.3a ). Immunostaining showed that GPR49/LGR5 is sufficiently expressed inan in vivo peripheral CEC (FIG. 3a ). It is worthy of special mentionthat, in in vitro cultured CEC (P0, P1), only minimum expression ofGPR49/LGR5 was recognized (FIG. 3a ). Real time PCR showed that theaverage GPR49/LGR5 mRNA expression is significantly downregulated in invitro CEC, as compared with expression in in vivo CEC (*p<0.05) (FIG. 3b). To the contrary, the expression revels of Nestin in both of in vivoand in vitro were the same (FIGS. 3a, b ).

Example 4 Expression of GPR49/LGR5 in Cultured Monkey CornealEndothelial Cell

A monkey corneal endothelial cell has a nature that the proliferationability in a living body is poor like human, but establishment of astable subculture method has been succeeded (Okumura et al., IOVS 2009,Vol. 50, No. 8, 3680-3687). Then, the amount of expression of GPR49/LGR5when a monkey corneal endothelial cell was subjected to subculture wasevaluated.

As a result of immunostaining, it was found that expression ofGPR49/LGR5 can be stably maintained in a corneal endothelial cellsubjected to subculture (FIG. 3c ). The amount of expression ofGPR49/LGR5 mRNA tended to be reduced by subculture as compared with amonkey corneal tissue, but the amount of expression could be maintained(FIG. 3d ).

In this way, a phase contrast microphotograph of monkey CEC showed thatboth of in vivo and in vitro (P0, P1) cells present a confluentmonolayer of a uniform hexagonal cell having a smaller size (FIG. 3c ).Immunostaining of these cells showed that GPR49/LGR5 is expressed to amoderate degree in both of in vivo and in vitro (FIG. 3c ), but the invitro average GPR49/LGR5 mRNA expression was gradually decreased, assubculture progressed (*p<0.05) (FIG. 3d ). In view of these findings,it seems that, when cells of human and monkey are used, GPR49/LGR5 canbe an important regulating factor for maintaining the undifferentiatedstate of in vitro CEC.

From the forgoing results, it is understood that GPR49/LGR5 possibly hasan important function in in vitro stable culture of a cornealendothelial cell.

Example 5 Study of Cell Biological Characteristic of GPR49/LGR5-PositiveCell

In order to study the cell biological characteristic of aGPR49/LGR5-positive corneal endothelial cell, analysis was performedusing FACS. Using a cultured monkey corneal endothelial cell which wasconfirmed to be able to be subjected to subculture in the state whereexpression of GPR49/LGR5 was maintained, the cell sizes and theproliferation abilities of a GPR49/LGR5-positive cell (GPR49/LGR5+) anda GPR49/LGR5-negative cell (GPR49/LGR5−) were compared and studied.

In order to investigate the characteristics of a GPR49/LGR5(+) cell anda GPR49/LGR5(−) cell, a subset of cells was isolated by flow cytometry.In order to inspect a procedure of cell sorting, expression at theprotein level was confirmed in a purified fraction by immunofluorescenceregarding GPR49/LGR5 (FIG. 4a ). Since the highest colony formingability is found in a minimum keratin-producing cell according to areport [Barrandon Y et al., Proc Natl Acad Sci USA, 1985; 82:5390-5394], the size of a cell in each of isolated fractions wasmeasured using the Scion Image software.

After cell sorting by FACS, when GPR49/LGR5+ and GPR49/LGR5− werephotographed with a fluorescent microscope and the size of each of 35cells was randomly measured by Image J (Wayne Rasband (NIH) freesoftware), it was found that GPR49/LGR5+ is significantly smaller ascompared with GPR49/LGR5− (FIG. 4a , FIG. 4b ). Then, it was found thatthe average size of a GPR49/LGR5(+) cell is significantly smaller thanthe average size of a GPR49/LGR5(−) cell (184.6±45.8 μm² vs. 326.78±78.8μm², N=35, **p<0.01).

Then, in order to evaluate the state of the cell cycle of each ofisolated cell fractions, an isolated cell fraction was cultured on acell chamber slide. The ratio of a Ki67-label cell in a GPR49/LGR5(+)cell and that in a GPR49/LGR5(−) cell were 14.2±3.87% and 0.58±0.5%,respectively, and the difference in a Ki67-label index was statisticallysignificant (*p<0.05)(FIG. 4c). As a result, it was revealed that theKi-67-positive cell rate is significantly higher in GPR49/LGR5+ (FIG. 4c). Then, in order to investigate the proliferation ability of each ofisolated cell fractions in further detail, FACS was employed concerningdouble staining of GPR49/LGR5 and Ki67, and in order to make arelationship between GPR49/LGR5 and cell proliferation more clear, acultured monkey corneal endothelial cell (passage number 3) was doublestained with anti-rabbit GPR49/LGR5 and anti-rabbit Ki-67, and analysisby FACS was tried. As a result, among all the cells, GPR49/LGR5+ was7.2%, and in particular, GPR49/LGR5+/Ki-67+ was 3.4% (FIG. 4d ). Inaddition, a cell of GPR49/LGR5−/Ki-67+ was not observed. FACS analysisshowed that while a GPR49/LGR5^(high)/Ki67^(high) cell fraction was3.4%, a GPR49/LGR5^(high)/Ki67^(low) cell fraction was 3.8% (FIG. 4d ).Most interestingly, all GPR49/LGR5^(low) cell fractions show the lowKi67 level (92.8%), and it is understood that CEC has no proliferationability without expression of GPR49/LGR5.

Example 6 GPR49/LGR5 as Target Gene of Hedgehog Signal

In the present Example, the function of GPR49/LGR5 in a Hedgehog signalwas investigated.

A Hedgehog signal was identified as a signal involved in morphosis at afetal stage, and thereafter, it was revealed that a Hedgehog signal isalso profoundly associated with a stem cell and tumorogenesis of anadult tissue. In addition, it has been reported that HH signaling has animportant role in various kinds of biological processes such asdifferentiation, proliferation and growth of a cell [Barker N et al.,Nature, 2007; 449: 1003-1007; Stanton B Z et al., MolBiosyst., 2010; 6:44-54; Tsuru T et al., Jpn J Ophthalmol., 1984; 28: 105-125]. As aligand of human, three kinds of proteins of SHH, Indian Hedgehog (Ihh),Desert Hedgehog (Dhh) have been identified. A twelve transmembraneprotein Patched1 (Ptch1) which is a receptor suppressively functioningto a signal, in the state where there is no ligand, suppresses cellmembrane localization of a seven transmembrane protein Smoothened (Smo),and also inhibits transmission to a downstream signal molecule. Underthis situation, a signal is not transmitted to a transcription factorGli family (Gli1, Gli2, Gli3) located downstream of Smo, andtranscription of a target gene does not occur. By binding of a ligand toPtch1, suppression of Ptch1 on Smo is lost, a signaling pathway from Smoto a transcription factor Gli family is activated, and a target genesuch as Cyclin D/E, Myc is transcribed. Both Ptch1 and Gli1 areassociated molecules of a signal, a target gene, and an index ofactivation of a signal.

As described above, GPR49/LGR5 has a high amount of expression in acorneal peripheral part (FIG. 2c , FIG. 2d , FIG. 2e ). Then, in orderto compare the amounts of expression of mRNA of Hedgehogsignal-associated molecules (Shh, Smo, Ptch1, Gli1, Gli2) in a humancorneal central endothelial cell and peripheral endothelial cell todefine the property of GPR49/LGR5 in CEC at the molecular level, first,expression of a HH signal transmission-associated molecule wasinvestigated.

As a result, it was observed that the amounts (levels of mRNA) ofexpression of Shh being a ligand, and transcription factors, Gli1 andGli2, were increased in CEC in a peripheral region, as compared with theamounts of expression in a central region, the amount of expression of aperipheral endothelial cell was elevated, and activation of a signal wasrecognized (FIG. 5a ). On the other hand, the expression levels ofSmoothened (Smo) and protein patched homolog 1 (Ptch1) which were each areceptor molecule of the HH pathway were the same, and no difference wasseen between such Ptch1 and Smo. Therefore, it was suggested that HHsignaling is clearly activated in CEC in a peripheral region and thereis variation depending on the place of the HH signaling activity.

In order to investigate a relationship between GPR49/LGR5 and a Hedgehogsignal, and determine whether expression of GPR49/LGR5 in CEC wasregulated by a HH signaling pathway or not, a relationship betweeninfluence of activation or suppression of a Hedgehog signal onGPR49/LGR5 expression, and cell proliferation was studied usingrecombinant human Sonic hedgehog (rhShh), Purmorphamine being an agonistof Smo (Sinha et al., Nat. Chem. Biol. 2: 29-30, 2006), and Cyclopaminebeing an antagonist (Chen et al., Genes Dev. 16 (21): 2743-2748, 2002).A human corneal tissue was treated with 100 ng/ml rhShh, 10 μMpurmorphamine, and 10 μM cyclopamine, and incubated under conditions of37° C. and 5% CO₂ for 3 days. A Descemet's membrane was peeled from acornea, and applied to a silane-coating slide, and GPR49/LGR5 and Ki-67were immunostained. In addition, RNA of a corneal endothelial cellincubated under the same conditions was extracted, and the amount ofexpression of mRNA was measured by real time PCR.

As a result of immunostaining, in expression of GPR49/LGR5, remarkableincrease (upregulation) was observed in both rhShh addition group andpurmorphamine addition group, as compared with a control (FIG. 5b ). Thenumber of GPR49/LGR5 expressing cells was increased, particularly, in arhShh addition group, and there was a tendency in a purmorphamineaddition group that a GPR49/LGR5-positive cell was strongly expressed.From this, it was presumed that while rhShh functions to activate asignal by binding to Ptch1, purmorphamine is activated by binding toSmo, and thus functions to activate a signal of a GPR49/LGR5 expressingcell. In addition, clear expression suppression was confirmed incyclopamine. The same result was also confirmed at the mRNA expressionlevel (FIG. 5c ). In addition, an expression pattern of Gli1 and Gli2was the same as that of GPR49/LGR5, but HH activation had no dramaticinfluence on a HH receptor (Ptch1) (FIG. 5c ).

In addition, in order to reveal whether the HH pathway induced CECproliferation in vivo or not, an immunohistochemical study on Ki67 wasperformed. Since human CEC is mitotically inactive, and exhibits a weakproliferation ability, or exhibits no proliferation ability in vivo[Joyce N C, Prog Retin Eye Res. 2003; 22: 359-389], a Ki-67-positivecell could not be detected in all groups (FIG. 5b ), and therefore, itis considered that promotion of cell proliferation by a Hedgehog signalin a human corneal tissue does not occur, and it is understood thatstimulation of only the HH pathway is insufficient for inducing in vivoCEC proliferation.

In order to inspect whether the same result was obtained in a culturedhuman corneal endothelial cell or not, a primary human cornealendothelial cell was seeded on an 8-well chamber slide, and 100 ng/mlrhShh, 2 μM purmorphamine, and 2 μM cyclopamine were added to a culturemedium, and thereafter, the resultant was incubated under conditions of37° C. and 5% CO₂. After 3 days, immunostain of GPR49/LGR5 and Ki-67 wasperformed. In addition, RNA of a corneal endothelial cell incubatedunder the same conditions was extracted, and the amount of expression ofmRNA was measured by real time PCR.

A cell expressing GPR49/LGR5 was confirmed only in a rhShh additiongroup (FIG. 5d , FIG. 5c ). There was no Ki-67-positive cell in acorneal tissue, but in a cultured corneal endothelial cell, the amountof expression was elevated as compared with a control, with respect to arhShh addition group and a purmorphamine addition group (FIG. 5f ). In acyclopamine addition group, abnormality of cell morphology andsuppression of cell proliferation were recognized.

CEC maintains the ability to proliferate in vitro, according to a report[Engelmann K et al., Invest Ophthalmol Vis Sci., 1988; 29: 1656-1662],and therefore, the present inventors studied whether the HH pathwayinduced in vitro proliferation of CEC or not, as described above.Expression of Ki67 was found to be upregulated in response to SHH andpurmorphamine stimulation, but this was not upregulated in response tocyclopamine (FIG. 5g ). These findings show that the HH pathway caninduce CEC proliferation under an in vitro situation. The presentinventors determined that CEC treated with cyclopamine could notmaintain a normal hexagonal form thereof (FIG. 5f ). In consideration ofthese findings, the present inventors first found that GPR49/LGR5 is atarget molecule of HH signaling in CEC, and maintenance of CEC isregulated partially by the HH pathway.

Example 7 Suppression of GPR49/LGR5 Gene Expression Using shRNA

In order to make clear a relationship between GPR49/LGR5 and a hedgehogsignal, suppression of GPR49/LGR5 expression by shRNA was carried outusing a cultured monkey corneal endothelial cell highly expressingGPR49/LGR5.

When the condition under which expression of GPR49/LGR5 mRNA wassignificantly decreased was first studied by real time PCR, it wasconfirmed that shGPR49/LGR5-589 can suppress the amount of expression ofGPR49/LGR5 mRNA by about 60% as compared with a control (FIG. 6a ).Then, in order to study the influence of suppression of expression ofGPR49/LGR5 on a Hedgehog signal, the amounts of expression of mRNA ofHedgehog signal-associated molecules (Ptch1, Gli1, Gli2) were analyzedusing a monkey corneal endothelial cell transfected withshGPR49/LGR5-589. As a result, no changes in the amounts of expressionof Hedgehog signal-associated molecules could be confirmed (FIG. 6b ).Without being bound to any theory, there is a possibility thatsuppression of expression of a PR49/LGR5 gene influenced proliferationability.

Example 8A Force Expression of GPR49/LGR5 Gene

In order to analyze the function of GPR49/LGR5 in a corneal endothelialcell, forced expression of GPR49/LGR5 by a gene introduction method wastried using a cultured human corneal endothelial cell in whichGPR49/LGR5 expression was remarkably reduced under normal cultureconditions.

In a cultured human corneal endothelial cell transfected with aGPR49/LGR5 expression vector, about 60 times of expression elevation wasrecognized as compared with a control (FIG. 6d ). In a cell with a geneof GPR49/LGR5 introduced therein, cell differentiation was suppressed,and expression of Na⁺/K⁺ ATPase used for evaluating the pumping functionof a corneal endothelial cell was elevated (FIG. 6c ). Using a cellunder this condition, the amounts of expression of mRNA of Hedgehogsignal-associated molecules (ptch1, gli1, gli2) were investigated. As aresult, the amount of expression was suppressed (negative feedback) inall the associated molecules (FIG. 6d ).

(Discussion)

From the above Example, it was made clear that GPR49/LGR5 isspecifically expressed in a stem cell and a precursor cell of a cornealendothelial cell existing in a peripheral part of a corneal tissue. Inaddition, it was made clear that a Hedgehog signal has the function ofpromoting proliferation of a corneal endothelial cell, and GPR49/LGR5being a downstream gene of a Hedgehog signal plays a particularlyimportant role in maintaining undifferentiation property of a cell bysuppressively functioning on a Hedgehog signal.

From the result of flow cytometry using a cultured monkey cornealendothelial cell, expression of a Ki-67-positive cell being a cellproliferation marker was observed only in a GPR49/LGR5-positive cell(FIG. 4c, d ). In a cultured human corneal endothelial cell, expressionof GPR49/LGR5 was elevated by activation of a Hedgehog signal (FIGS. 5d,e ), and promotion of cell proliferation was confirmed (FIGS. 5f, g ).Since the occurrence of cell proliferation by activation of a Hedgehogsignal has been reported by promotion of proliferation of an adultneural stem cell by addition of rhShh (Lai et al., Nature neuroscience6: 21-27, 2003), and promotion of proliferation and differentiation of astromal stem cell by addition of purmorphamine (Wu et al., Chem. Biol.11, 1,229-1,230, 2004), it was considered that a corneal endothelialcell similarly has the cell proliferation promoting action by activationof a signal. Since a stable method for culture of a corneal endothelialcell is not currently established, it is understood that the presentinvention can be possibly applied to establishment of a novel culturemethod utilizing the cell proliferation promoting effect by Hedgehogsignal activation. It is understood that, in the present Example, byactivation of SHH, proliferation is elevated in a cultured endothelialcell. In addition, it is understood that SHH can be used as a marker ofthe degree of differentiation. In contrast, in a corneal endothelialtissue, by activation of a Hedgehog signal, expression of GPR49/LGR5 waselevated, but the effect of promoting cell proliferation was notobtained (FIG. 5b ). It has been reported that human corneal endothelialcells in a living body are extremely close to each other, and unlessadhesion between cells is alleviated using EDTA, the cell cycle does notwork (Senoo et al., IOVS 41 2930-2935, 2000). Therefore, it was presumedthat expression of a target gene is elevated by activation of a Hedgehogsignal, but adhesion between cells in a living body is very intimate tonot lead to cell proliferation. In addition, by an experiment of forcedexpression of GPR49/LGR5, the present inventors paid an attention tothat expression of Ptch1, Gli1 and Gli2 which were each an associatedmolecule of a Hedgehog signal and also an index of signal activation wasreduced (FIG. 6d ). That is, there is a possibility that, althoughGPR49/LGR5 exists as a target gene of a Hedgehog signal, it has a roleas a negative control factor which suppresses expression of associatedmolecules of a Hedgehog signal. Therefore, it is presumed thatexpression of a cell proliferation-associated gene such as Cyclin D/E orMyc which is other target gene is also suppressed by suppression of aHedgehog signal. Further, GPR49/LGR5 forced-expressed cells tend to bein contact with each other at a high density, and the function of acorneal endothelial cell is high. It was presumed that controlling ofprogression into proliferation and differentiation due to Hedgehogsignal activation by GPR49/LGR5 contributes to maintenance ofundifferentiation property. There is a possibility that GPR49/LGR5 isassociated with adhesion between cells, and it is considered thatfurther research is necessary as a study theme in the future.

(Summary)

In summary, there is a possibility that cell proliferation of a cornealendothelial cell in a living body is controlled by a Hedgehog signalwith Sonic hedgehog as a ligand. In addition, it is considered thatGPR49/LGR5 being a target gene of a Hedgehog signal is involved in theundifferentiation property-maintaining mechanism of a cornealendothelial cell.

Example 8B Further Analysis of GPR49/LGR5 (Downregulation of GPR49/LGR5Decreased Proliferation of CEC)

The direct action of GPR49/LGR5 on CEC was revealed by knockdown ofGPR49/LGR5 by shRNA. Due to the fact that cultured human CEC expressesGPR49/LGR5 rarely (FIGS. 3a, b ), cultured CEC of a primate was employedin this experiment. Nine sets of shRNA were designed, and validity ofthe knocking down ability thereof was studied. It was found thatshRNA-589 among them is most effective in knocking down GPR49/LGR5 mRNAexpression (about 60% knockdown) (FIG. 6A-A). Real time PCR with respectto Ptch1, Gli1 and Gli2 showed that a significant difference is not seenbetween a shLGR5 group and a control (FIG. 6A-A). In order to verifyinfluence of GPR49/LGR5 gene knockdown on CEC proliferation,immunocytochemical study with respect to Ki67 was conducted. As comparedwith a control, cell morphology of a shLGR5-treated cell did not changedramatically, but the number of Ki67(+) cells in the shLGR5-treated cellwas greatly decreased (FIG. 6A-B). These findings showed thatdownregulation of GPR49/LGR5 has no influence on the HH pathway, butdecreases in vitro CEC proliferation.

(Permanent GPR49/LGR5 Expression Inhibited Mesenchymal Transition (MT)via Wnt Pathway)

In order to investigate the direct action of permanent GPR49/LGR5expression on CEC, the present inventors tried to allow GPR49/LGR5 to beoverexpressed using a lentivirus containing CMV-LGR5-mRFP. In thisexperiment, human cultured CEC was employed. This is because itexpresses GPR49/LGR5 rarely (FIG. 3a ). Real time PCR showed thatexpression of GPR49/LGR5 in a cell transfected with GPR49/LGR5 is about60 times higher than expression in a cell transfected with a NT vector(FIG. 6B-B). When an immunofluorescence method was used, it wasconfirmed that expression of GPR49/LGR5 in a cell transfected withGPR49/LGR5 is increased as compared with expression in a NT cell (FIG.6B-A). Very interestingly, the relative mRNA level of a HH signalingmolecule in a cell transfected with GPR49/LGR5 is downregulated ascompared with that in a NT cell (FIG. 6B-B), and it is understood thatGPR49/LGR5 functions as a negative feedback regulation factor of the HHpathway.

Human CEC easily undergoes fibroblast-like morphological change undernormal culture conditions, according to a report [Peh G S et al.,Transplantation., 2011; 91: 811-819]. After transfection with alentivirus, NT cells showed an enlarged elongate form (fibroblast-likechange), and the shape was not a uniform hexagonal shape (FIG. 6B-A).Very interestingly, it was shown that a cell transfected with GPR49/LGR5is gradually changed in terms of its morphology and becomes a compactuniform hexagonal cell having a smaller size, and takes normalphysiological morphology again (FIG. 6B-A). The cell density of a celltransfected with GPR49/LGR5 was greatly increased as compared with thatof a NT cell (FIG. 6B-C). In order to investigate the function ofcultured CEC transfected with NT and LGR5 vectors, animmunohistochemical method was performed with respect to Na⁺/K⁺ ATPaseand ZO1. It was found that these two functional proteins areconsiderably highly expressed in a cell transfected with GPR49/LGR5 thanin a NT cell (FIG. 6B-A). In consideration of these findings, it seemsthat GPR49/LGR5 can be an important molecule for maintaining a normalCEC phenotype.

From very interesting finding observed in a cell transfected withGPR49/LGR5, the present inventors further studied whether permanentexpression of GPR49/LGR5 can block a MT process or not. The expressionlevels of epithelium NT (EMT)-associated molecules (Snail, Slug, Twistand collagen 1) [Lee J M et al., J Cell Biol., 2006; 172: 973-981] wereinvestigated using real time PCR. Most importantly, the relative mRNAlevels of EMT markers except for Slug were lower in a cell transfectedwith GPR49/LGR5 than in a NT cell (FIG. 6B-D), and it is understood thatpermanent GPR19/LGR5 expression blocked a MT process. The presentinventors further investigated which pathway regulated endothelial MTobserved in CEC. Recent research suggests that a Wnt/β-catenin signalingpathway plays an important role in EMT [Lee J M et al., J Cell Biol.,2006; 172: 973-981]. For this reason, using Western Blotting analysis,the expression level of a Wnt/β-catenin-associated molecule wasinvestigated. It is worthy of special mentioning that the protein levelsof cytosol (non-membrane-bound type) β-catenin and phosphorylation LDLreceptor-associated protein 6 (p-LRP6) were greatly decreased in a celltransfected with GPR49/LGR5 (FIG. 6B-F). These findings showed thatpermanent GPR49/LGR5 expression inhibits corneal endothelial MT througha Wnt/β-catenin pathway.

(RSPO1 Accelerated CEC Proliferation and Inhibited MT Through WntPathway.)

GPR49/LGR5 was an orphan receptor of a G protein-coupled receptorsuperfamily, and a ligand thereof was not known previously. However,some reports in recent years have demonstrated that RSPO functions as aligand of GPR49/LGR5, and regulates Wnt/β-catenin signaling [Carmon K Set al., Proc Natl Acad Sci USA., 2011; 108: 11452-11457; de Lau W etal., Nature, 2011; 476: 293-297; Glinka A et al., EMBO Rep., 2011; 12:1055-1061]. Interestingly, the present inventors found that RSPO1, 2, 3and 4 are expressed in cells of an epithelium, a parenchyma and anendothelium of a cornea, and RSPO1, 2 and 3 are expressed only in CEC ina peripheral region (FIG. 6C-A). In order to determine the function ofRSPO on CEC differentiation, the present inventors cultured primate CECwith human recombinant RSPO, or without human recombinant RSPO. It isworthy of special mention that only cultured human CEC treated withRSPO1 showed a compact and uniform hexagonal cell having a smaller size,and on the other hand, other RSPOs had no influence on in vitro CECdifferentiation (FIG. 6C-B). In order to determine the function of RSPOon CEC proliferation, the present inventors performedimmunohistochemical research on Ki67. Most surprisingly and veryinterestingly, CEC incubated with RSPO1 showed a dramatically increasedlevel of the Ki67(+) cell ratio as compared with other RSPOs (FIG.6C-C). In view of these findings, the present inventors considered thatamong a RSPO family, particularly, RSPO1 can play an important role inmaintenance of CEC.

Finally, in order to further determine influence of RSPO1 on CEC, thepresent inventors maintained a secondary culture of human CEC in thepresence or absence of RSPO1. Through culturing of CEC under both theconditions, the present inventors clearly observed that while a cellcultured with RSPO1 maintained its hexagonal form, a cell culturedwithout RSPO1 showed a fibroblast-like phenotype (FIG. 6B-E). The celldensity of a RSPO1-treated cell increased as compared with that of anon-treated cell (FIG. 6B-E). In order to verify which pathway regulatedthis type of corneal endothelial MT, the present inventors investigatedthe expression level of a Wnt/β-catenin-associated molecule usingWestern blotting analysis. Surprisingly, the protein level of cytosolβ-catenin and p-LRP6 in a cell transfected with GPR49/LGR5 and treatedwith RSPO1 clearly decreased as compared with that of a NT cell.Further, the protein level of a cell transfected with NT and GPR49/LGR5and treated with RSPO1 decreased as compared with that of a cell groupnot treated with RSPO1 (FIG. 6B-F). These results suggested thatstimulation of a cell overexpressing GPR49/LGR5 with RSPO1 acceleratesdegradation of pLRP and turnover of β-catenin.

(Discussion)

Since a majority of mammals gains a majority of external informationthrough a cornea, a corneal tissue is extremely important. In recentyears, from the fact that cornea transplant technique has undergoneparadigm shift from keratoplasty to corneal endothelium transplantation,CEC has particularly attracted attention. For this reason, in order toscientifically and clinically establish novel therapy of the nextgeneration for treating cornea-associated blindness in the world, it isconsiderably important to understand the molecular mechanism of acorneal endothelial stem cell/precursor cell. However, with respect tothe molecular mechanism of them, only little is currently known.

It has been reported that the characteristics and the proliferationability of CEC are different between CEC positioned in a central regionof a cornea, and CEC positioned in a peripheral region of a cornea[Bednarz J et al., In vitro Cell Dev Biol Anim., 1998; 34: 149-153], andit has been shown by research that a cornea has a higher endothelialcell density in a peripheral region than in a central region[Schimmelpfennig B H, Invest Ophthalmol Vis Sci., 1984; 25: 223-229].Further, CEC derived from a peripheral region maintains a higherreplicating ability than that of CEC derived from a central region,according to a report [Mimura T et al., Invest Ophthalmol Vis Sci.,2006; 47: 1387-1396], and CEC in a peripheral region contains a largenumber of precursor cells, and has a stronger self-renewal ability thanCEC in a central region [Mimura T et al., Invest Ophthalmol Vis Sci.,2005; 46: 3645-3648]. Therefore, there is a high possibility that ahuman corneal endothelial stem cell/precursor cell are distributedmainly in a peripheral region. In fact, a stem cell/precursor cellmarker concerning CEC has not been previously revealed. The result ofthe present research first verified that CEC exhibits regional diversityregarding GPR49/LGR5 expression. When these findings and an especialexpression pattern of GPR49/LGR5 are considered, there is a possibilitythat this serve as a first marker concerning a population including acorneal endothelial stem cell.

It has been reported that a keratin-producing cell stem cell can beidentified from a temporarily proliferating cell or a differentiatedcell [Barrandon Y et al., Proc Natl Acad Sci USA, 1985; 82: 5390-5394].In an epidermis, a response to a phorbol ester of the minimum ofkeratin-producing cells is different from that of other cells. Thesekeratin-producing cells also exhibited the highest colony formingability. CEC was different from that of a keratin-producing cell derivedfrom an ectoderm, and the average diameter of a GPR49/LGR5(+) cell bythe present inventors was, in fact, smaller than that of a GPR49/LGR5(−)cell. Based on these findings, and the size of peripheral CEC reported,it seems that the size of a cell serves as a latent index of a corneaendothelial stem cell/precursor cell.

The present inventors have found that GPR49/LGR5 is an importantmolecule for maintenance of the undifferentiated state of CEC, and invitro regulation of a normal cell phenotype. The present inventors havealso found that an isolated cell fractionated based on the GPR49/LGR5expression intensity can generate different cell populations havingdifferent properties. Only a cell in a GPR49/LGR5(−) populationexhibited an exceptionally high proliferation ability which was thecharacteristic associated with a stem cell/precursor cell population.Based on these findings, a peculiar expression pattern andunavoidability under the in vitro condition, there is a possibility thatthere is some relationship between GPR49/LGR5 and the function of acorneal endothelial stem cell/precursor cell.

The previous research has shown that SSH in a high concentration resultsin a remarkable increase in retinal precursor cell population, and thetotal increase in accumulation of a differentiated cell [Stanton B Z etal., Mol Biosyst., 2010; 6: 44-54]. The finding of the presentapplication shows that, under the in vitro situation, a HH pathway caninduce proliferation of CEC, in agreement with the previous report. HHis a family of a secretory molecule which functions as a morphogenduring a plurality of aspects of generation of a wide range of tissuetypes. HH regulates proliferation and living of a cell to be involved indetermination of left and right asymmetry and determination of anantero-posterior axis in determination of a limb pattern. In CEC, thereis regional variation in the HH signal activity, and based on thefindings by the preset inventors, there is a possibility that HHsignaling controls corneal endothelial morphosis.

RSPO is a family of a 4 cysteine-rich secretory protein, isolated as astrong enhancing substance of Wnt/β-catenin signaling. A large amount ofinformation regarding the cell biological function of RSPO has beenrevealed, particularly regarding a role of an orphan receptor LGR4/5/6as a ligand, over past several years. From these updated importantfindings, the present inventors further studied whether RSPO couldinfluence the function of human CEC or not. Since human CEC ismitotically inactive, and has substantially no regeneration ability invivo, compensating extension of a remaining endothelial cell isgenerated after loss of a corneal epithelium due to a disease or trauma.As far as the present inventors know, there is no report regarding auseful induction reagent or molecule which increases proliferation ofhuman CEC and the level of the CEC density. The finding of the presentresearch first showed that CEC incubated with RSPO1 exhibits adramatically increased level of cell proliferation and the cell density,and it is suggested that there is a possibility that this moleculeserves as a first candidate molecule for reconstituting a corneal whichhas received damage by local administration or a culture reagent.

Some research have suggested that a Wnt/β-catenin pathway plays animportant role in EMT, and Wnt/β-catenin-dependent signaling regulatesexpression of an EMT-associated gene [Lee J M et al., J Cell Biol.,2006; 172: 973-981]. However, the previous reports have shown that RSPO,in fact, enhances Wnt/β-catenin signaling by functioning as a ligand ofGPR49/LGR5 [Carmon K S et al, Proc Natl Acad Sci USA., 2011; 108:11452-11457; de Lau W et al., Nature, 2011; 476: 293-297; Glinka A etal., EMBO Rep., 2011; 12: 1055-1061]. The actual mechanism regardingthis activation has unknown yet, and there are some contradictoryfindings, concerning whether GPR49/LGR5 is a positive regulatory factoror a negative regulatory factor of the Wnt pathway [Garcia M I et al.,DevBiol., 2009; 331: 58-67; Schuijers J et al., EMBOJ., 2012; Walker Fet al., PLoSOne., 2011; 6: e22733]. One possible explanation is that themolecular mechanism depends on a tissue, an organ and a species thereof.A cornea is a peculiar blood vessel-free tissue which is maintained bytear and aqueous humor. To the contrary, a majority of other organs aremaintained by supporting of a blood vessel structure, and it issuggested that the characteristics and the mechanism of a corneal cellare fundamentally different from those of an epithelial cell of othertissues. Therefore, based on the findings of the present research, RSPO1dramatically accelerates proliferation of CEC, and inhibits cornealendothelial MT through the Wnt pathway.

(Conclusion)

As conclusion, the findings of the present research first verify thefunction of GPR49/LGR5 in human CEC (FIG. 6D). It was demonstrated thatGPR49/LGR5 serves as a powerful tool upon identification of many stemcell/precursor cell populations. By regulation of GPR49/LGR5 through HHand Wnt pathways, completeness of CEC was sufficiently systemized, andmaintained. In addition, it is understood that RSPO1 being a GPR49/LGR5ligand can develop a novel sufficient protocol for providing aneffective increase in CEC, and RSPO1-based three-dimensional culture ormedical treatment promises the future regenerative medicine not only fortreatment of corneal dysfunction, but also treatment of a variety ofserious systemic diseases.

Example 9 Cell Proliferation Promoting Effect of R-Spondins

In the present Example, an experiment was performed for the purpose ofstudying the cell proliferation promoting effect of R-spondins,particularly R-spondin 1 in culture of a corneal endothelial cell.

(Method)

After addition of an R-spondin 1 protein to a cultured monkey cornealendothelial cell and culturing for 48 hours, a cell during proliferationwas detected using the Click-iT Edu imaging Kit, and the positivityrates of EdU and the cell densities of a control cell and a cell towhich R-spondin 1 was added were compared.

EdU (5-ethynyl-2′-deoxyuridine) is a modified nucleic acid taken intoDNA at a DNA synthesis phase by a chemical reaction, and is widely usedin place of a conventional BrdU for identifying a DNA synthesis phasecell. By measuring the positivity rate of EdU, the proliferative cellrate in a cultured cell is found.

(Reagents Used)

-   Click-iT EdU Imaging Kit (Invitrogen Cat. C10337)-   Recombinant Human R-spondin 1 (R&D Cat. 4645-RS)

(Cell Used)

-   Cultured monkey corneal endothelial cell (Lot. 20111222-4 P2)-   (Experiment: Study of cell proliferation promoting effect of    R-spondin 1 in cultured monkey corneal endothelial cell culture)

(Procedure) (1) Seeding of Cultured Monkey Corneal Endothelial Cell andAddition of R-Spondin 1

A cultured monkey corneal endothelial cell (Lot. 20111222-4 P2) wastreated with 0.05% trypsin at 37° C. for 10 minutes, peeled from a T-25flask, and suspended in a culture medium (10% FBS+bFGF/DMEM). The numberof cells was counted, and the suspension was adjusted with a culturemedium to 3×10⁴ cells/300 μl. Cells were seeded on a Lab-TekII chamberSlide (8 well) coated with FNS, in 300 μl/well.

(2) Detection of Proliferating Cell Using Click-iT EdU Imaging Kit

At 7 days after cell seeding, a medium was exchanged with a mediumcontaining R-spondin 1 (0, 10, 50 ng/ml) in the state where cells werealmost confluent, and at 24 hours after addition of R-spondin 1,Click-iT EdU was added so that the final concentration was 10 μM.(Although a manual of Kit recommended that a half of a medium wasexchanged, a medium was not exchanged) After additional 24 hours (48hours after addition of R-spondin 1), cells were fixed with 4% PFA/PBS,EdU was detected according to a manual of Kit, and the EdU-positive cellrate was counted (FIGS. 8 and 9).

(3) Measurement of Cell Density

A chamber slide after staining was photographed with a phase contrastmicroscope, and the cell density was measured using the cornealendothelial cell density calculating software, Konan Storage SystemKSS-400EB (FIG. 10).

(Result)

As a result of counting of EdU-positive cell/DAPI, an EdU-positive cellwas found to be increased twice in a well to which 10 ng/ml or 50 ng/mlR-spondin 1 was added, as compared with a control. In addition, as aresult of measurement of the cell density, the cell density was found tobe increased 1.3 times in a well to which 10 ng/ml or 50 ng/ml R-spondin1 was added, as compared with a control.

(Discussion)

The cell proliferation promoting effect of R-spondin 1 was recognized ina cultured monkey corneal endothelial cell. In addition, the same effectis expected to be exerted in a cultured human corneal endothelial celland a human corneal endothelial tissue.

Example 10 Cell Proliferation Promoting Effect of R-Spondins in CulturedHuman Corneal Endothelial Cell

In order to investigate a relationship between R-spondin 1 (RSPO1),R-spondin 2 (RSPO2), R-spondin 3 (RSPO3), or R-spondin 4 (RSPO4) anddifferentiation of a corneal endothelial cell, the relationship wasstudied using RSPO1. Reagents used are as follows.

-   RSPO1 R&D systems catalog No.: 4645-RS-   RSPO2 R&D systems catalog No.: 3266-RS-   RSPO3 R&D systems catalog No.: 3500-RS-   RSPO4 R&D systems catalog No.: 4575-RS

A cultured human corneal endothelial cell was cultured for 7 days underconditions of 37° C. and 5% CO₂, with being divided into a group ofaddition of RSPO1 (50 ng/ml) and a group of addition of no RSPO1. As aresult, morphology of a cell was maintained in a cobblestone manner inthe RSPO1 addition group, while a cell was differentiated intofibroblast-like cells in the non-addition group. From the above result,the effect of suppressing differentiation of a corneal endothelial cellwas recognized in RSPO1.

Then, for the purpose of investigating the influence of RSPO on cellproliferation, RSPO1 to 4 were incubated into a human cornealendothelial cell under conditions of 37° C. and 5% CO₂ for 1 day.Thereafter, Ki-67 being a cell proliferation marker was immunostained.As a result, a proliferation tendency was seen in all RSPO1 to 4, butonly RSPO1 statistically significantly promoted proliferation of a humancorneal endothelial cell.

Example 11 Effect of R-Spondins on Confluent Cell

In the present Example, whether R-spondins had the proliferation effecton a corneal endothelial cell which reached the confluent state or notwas confirmed.

(Method)

(Source and culture method): As a human corneal endothelial cell, acorneal endothelial cell was mechanically peeled together with a basalmembrane from a cornea for research, purchased from Seattle Eye Bank,and the corneal endothelial cell was peeled and recovered from the basalmembrane using collagenase (ROCHE catalog No.: 10 103 586 001), and wassubjected to primary culturing. As a medium, for human, Opti-MEM IReduced-Serum Medium, Liquid (INVITROGEN catalog No.: 31985-070), 8%fetal bovine serum (FBS)(BIOWEST, catalog No.: S1820-500), 200 mg/mlCaCl₂.2H₂O (SIGMA catalog No.: C7902-500G), 0.08% chondroitin sulfate(SIGMA catalog No.: C9819-5G), 20 μg/ml ascorbic acid (SIGMA catalogNo.: A4544-25G), 50 μg/ml gentamicin (INVITROGEN catalog No.: 15710-064)and 5 ng/ml EGF (INVITROGEN catalog No.: PHG0311), acclimated for a 3T3feeder cell, were used.

After subculturing, at the time point where cells reached confluent andwere cultured for 2 weeks or longer, and no clear change in the cornealendothelium density was recognized, R-spondin 1 was added into a mediumin a concentration of 10 ng/ml, and culture was continued. Observationof cell morphology was performed with a phase contrast microscope, andthe cell density was calculated.

(Result)

The results are shown in FIG. 15. The average corneal endothelial celldensity at the time point where cells reached confluent and no clearchange in the corneal endothelial density was recognized was 566.8cells/mm², and the density was increased with time, reached about 695cells/mm² on 7^(th) day, reached about 875 cells/mm² after 14 days, andreached 995.8 cells/mm² after 21 days. On the other hand, when a cell inthe same lot was cultured as a control in a medium not containingR-spondin 1 for 21 days being the same period, the density was 535.4cells/mm².

(Discussion)

These results mean that, in a corneal endothelial cell, the density ofwhich is easily decreased by culturing, the corneal endothelial celldensity can be increased by culturing using R-spondin 1. This means thatcells having a high corneal endothelium density being an importantprognosis determinant after transplantation can be transplanted, towardsclinical application of cultured corneal endothelial transplantation. Inaddition to usefulness for regenerative medicine of a cornealendothelium, it is understood that, also in other cells, use ofR-spondin 1 allows for transplantation of cells having a higherfunction.

Example 12 Production of Tissue in Which Corneal Endothelium Density isIncreased by R-Spondins

In the present Example, it is demonstrated that a tissue having anincreased corneal endothelium density can be prepared by treatment of acorneal tissue with R-Spondins.

(Method)

Eyeballs of white rabbit (Nacalai) euthanized for another object werepurchased, and a sclerocorneal section was prepared. The sclerocornealsection was cultured in an incubator at 37° C. for 1 week in DMEM(INVITROGEN, catalog No.: 12320) and 10% fetal bovine serum (FBS)(BIOWEST, catalog No.: S1820-500). After one week, the sclerocornealsection was fixed with 4% paraformaldehyde at room temperature for 10minutes, and after fixing, Ki67 (Sigma-Aldrich Co., catalog No.: P6834)was used as a marker of cell proliferation to immunostaining a cornealendothelial cell, and this was observed with a fluorescent microscope. Acorneal endothelial cell was subjected to nuclear staining with DAPI,and the Ki67-positive cell rate was calculated. In addition, a cornealendothelial cell was immunostained with ZO-1, and the cell density wascalculated.

(Result)

The results are shown in FIG. 16. When the culturing was performed in amedium containing R-spondin 1, a Ki67-positive cell was recognized at ahigher ratio as compared with a control, also in the sclerocornealsection. That is, while the rate of a Ki67-positive cell was about 1.0%in a control to which R-spondin 1 was not added, the rate was 4.49% inaddition of 1 ng/ml R-spondin 1, was 10.58% in addition of 10 ng/mlR-spondin 1, and was 7.94% in addition of 100 ng/ml R-spondin 1.

Further, the corneal endothelial cell density showed a significantlyhigh value by R-spondin 1. That is, the corneal endothelial cell densitywas 3674 cells/mm² in a control to which R-spondin 1 was not added, andwas increased to 4314 cells/mm² in addition of 1 ng/ml R-spondin 1,increased to 4626 cells/mm² in addition of 10 ng/ml R-spondin 1, andincreased to 5037 cells/mm² in addition of 100 ng/ml R-spondin 1.

(Discussion)

The result means that a tissue having an increased corneal endotheliumdensity being an important prognosis determinant after transplantationcan be transplanted by allowing R-spondin 1 to act on a corneal tissue,in corneal transplant medicine. In addition to usefulness in cornealtransplant medicine, it is understood that, also in other tissues, useof R-spondin 1 allows for transplantation of a tissue having a higherfunction.

Example 13 Proliferation Effect in Cells of Corneal Parenchyma,Epithelium, Retinal Pigment Epithelium (RPE), Vitreous Body, and theLike

In the present Example, the proliferation effects of R-spondins in cellsof a corneal parenchyma, an epithelium, RPE, a vitreous body and thelike are confirmed, respectively. Culturing methods are listed below. Ineach culturing method, by culturing in the presence or absence ofR-spondins according to the above Example, it can be confirmed that theeffect on proliferation is promoted.

(Corneal Parenchymal Cell Culture Method)

A corneal parenchymal cell is cultured based on the procedure ofYamamoto M, Quantock A J, Young R D, Okumura N, Ueno M, Sakamoto Y,Kinoshita S, Koizumi N. Mol Vis. 2012; 18: 1727-39. In brief, a rabbitcornea is incubated with 1.2 U/ml Dispase (Invitrogen) at 37° C. for 1hour. Thereafter, a corneal epithelium and a corneal endothelium areremoved by mechanical scraping. Then, a corneal parenchyma is cut intoabout 1 cm² sections, and these are incubated in DMEM/F12 containing 1mg/ml collagenase A (Roche Diagnostics Japan) and 1%penicillin-streptomycin at 37° C. overnight. After centrifugation at1500 rpm (440×g) for 3 minutes, cells are successively cultured in acell-free medium (DMEM/F12 containing 10 μg/ml 1 mM ascorbic acid, and1% penicillin-streptomycin) for 48 hours. Cells thus obtained can beused to perform an experiment.

(Retinal Pigment Epithelium (RPE) Cell Culture Method)

RPE cells are cultured based on the procedure of Hatanaka H, Koizumi N,et al., Investigative Ophthalmology & Visual Science. 2012; 53(11):6955-6963. In brief, a monkey retinal pigment epithelial cell (MRPEC) iscultured from a rear region of eyeballs taken out from cynomolgus monkey(3 to 5 years old, corresponding to 5 to 20 years old in human; obtainedfrom Nissei Bilis Co. Ltd., Otsu Japan). Then, a RPEC fragment of MRPEC(cell mass of MRPEC) is separated and prepared based on the procedure(Maminishkis A, Chen S, Jalickee S, et al. Confluent monolayers ofcultured human fetal retinal pigment epithelium exhibit morphology andphysiology of native tissue. Invest Ophthalmol Vis Sci. 2006; 47:3612-3624) previously reported regarding a human fetal RPE. Then, MRPECis cultured, in DMEM/F12 supplemented with 10% FBA, 50 U/ml penicillinand 50 μg/ml streptomycin, on a dish coated with FNC Coating Mix(registered trademark), and further expanded at 37° C. under ahumidified environment in 5% CO₂. Then, a culture medium is exchangedevery 2 days. When cells reach confluent in 5 to 7 days, cells arerinsed with the Dulbecco's phosphate buffered physiological saline (PBS)not containing Ca²⁺ and Mg²⁺, trypsin-treated with 0.05% trypsin-EDTA(Life Technologies) at 37° C. for 5 minutes, and subjected to subcultureat a ratio of 1:2 to 4. Cultured MRPEC at 1 to 3 passages is used in allexperiments.

(Corneal Epithelial Cell Culture Method)

An amnion was collected according to the method approved by EthicsCommittee of Kyoto Prefectural University of medicine, and used forresearch. After written consent was obtained from a pregnant womanscheduled to receive Caesarean section, having neither an infectiousdisease nor complication, an amnion was sterilely collected in Caesareansection, washed, immersed in a 50% glycerol DMEM solution, and frozenand preserved at −80° C. Then, a corneal epithelial stem cell collectedfrom a human sclerocorneal tissue for which authorization was gained touse for the research purpose from Northwest Lion Eye Bank (Seattle,Wash., USA) was cultured. In order to prepare a cultured cornealepithelial sheet containing a corneal epithelial stem cell, a cornealepithelial cell was recovered as a cell suspension by enzyme treatmentusing 1.2 U Dispase at 37° C. for 1 hour. Then, the cell suspension wasseeded on an amnion, and cultured in an incubator at 37° C. in 5% CO₂for about 2 weeks.

(Vitreous Body Cell (Hyalocyte) Culture Method)

A vitreous body cell is cultured in accordance with the method reportedin Sommer F, Pollonger K, et al. Graefes Arch Clin Exp Ophthalmol, 2008.First, a vitreous body is collected from monkey eyeballs, and washedwith DMEM with 1% penicillin/streptomycin added thereto three times. Thesample is immersed in 1 mg/ml collagenase, and incubated overnight at37° C. with rotations. After centrifugation, the supernatant is removed,and the resultant is washed with DMEM with 1% penicillin/streptomycinadded thereto. DMEM, 10% FBS, and 1% P/S are added, and the resultant isseeded on a culture dish.

Example 14 Experiment Concerning Other Cells

In the present Example, concerning a nerve cell, a conjunctivalepithelium, an amniotic epithelium, an oral mucosa epithelium, and anose mucosa epithelium, the proliferation effect of R-spondins isconfirmed, respectively. A culture method can be performed in accordancewith a known method. In each culture method, by culturing in thepresence or absence of R-spondins in accordance with the above Examples,it can be confirmed that the effect on proliferation is promoted.

Example 15 Preparation Example: Cornea Preservation Solution ContainingAgent for Stimulating Proliferation or Controlling Differentiation

In the present Example, as Preparation Example, the cornea preservationsolution containing a culture-normalizing agent of the present inventionis produced as follows.

According to a conventional method, the following preservation solutionis prepared.

-   R-spondin 1 10-500 ng/ml (appropriately adjusted)-   Optisol-GS (Bausch-Lomb) proper quantity-   Total amount 100 mL

Each ingredient can be obtained from R&D Systems Inc. (Minneapolis,Minn.).

Example 16 Preparation Example of Infusion Preparation Example of EyeDrops

The composition of each test substance in each concentration is shownbelow.

R-spondin 1 10-500 ng/ml (appropriately adjusted) Sodium chloride  0.85g Sodium dihydrogen phosphate dihydrate  0.1 g Benzalkonium chloride0.005 g Sodium hydroxide proper quantity Purified water proper quantityTotal amount 100 mg (pH 7.0)

Eye drops can also be diluted with a base.

The composition of a base is as follows.

Sodium chloride  0.85 g Sodium dihydrogen phosphate dihydrate  0.1 gBenzalkonium chloride 0.005 g Sodium hydroxide proper quantity Purifiedwater proper quantity Total amount 100 mg (pH 7.0)

Each ingredient can be obtained from R&D Systems Inc. (Minneapolis,Minn.).

As described above, the present invention has been exemplified usingpreferable embodiments of the present invention, but it is understoodthat the scope of the present invention should be construed only by thescope of claims. It is understood that patents, patent applications andliteratures cited herein are incorporated herein as reference, as if thecontents thereof themselves are specifically described herein.

INDUSTRIAL APPLICABILITY

A differentiation marker and a differentiation controlling technique ofan eye cell are provided, and a technique which can be utilized inindustries involved in a technique associated with corneal transplant(cell culture industry, pharmacy, and the like) is provided.

SEQUENCE LISTING FREE TEXT

-   SEQ ID No.: 1: Gene sequence encoding human GPR49/LGR5 (OMIM:    606667; NM_003667.2)-   SEQ ID No.: 2: Amino acid sequence of human GPR49/LGR5 (OMIM:    606667; NP_003658.1)-   SEQ ID No.: 3: Gene sequence encoding R-spondin 1 (RSPO1) (OMIM:    609595; NM_001038633) (transcript variant 1)-   SEQ ID No.: 4: Amino acid sequence of R-spondin 1 (RSPO1) (OMIM:    609595; NM_001038633) (transcript variant 1)-   SEQ ID No.: 5: Gene sequence encoding R-spondin 2 (RSPO2) (OMIM:    610575; NM_178565)-   SEQ ID No.: 6: Amino acid sequence of R-spondin 2 (RSPO2) (OMIM:    610575; NM_178565)-   SEQ ID No. 7: Gene sequence encoding R-spondin 3 (RSPO3) (OMIM:    610574; NM_032784)-   SEQ ID No.: 8: Amino acid sequence of R-spondin 3 (RSPO3) (OMIM:    610574; NM_032784)-   SEQ ID No.: 9: Gene sequence encoding R-spondin 4 (RSPO4) (OMIM:    610573; NM_001029871) (transcript variant 1)-   SEQ ID No.: 10: Amino acid sequence of R-spondin 4 (RSPO4) (OMIM:    610573; NM_001029871) (transcript variant 1)-   SEQ ID No.: 11: Gene sequence encoding SHH (SONIC HEDGEHOG) (OMIM:    600725; NM_000193)-   SEQ ID No.: 12: Amino acid sequence of SHH (SONIC HEDGEHOG) (OMIM:    600725; NM_000193)-   SEQ ID No.: 13: Forward primer of HumanGPR49 (Table 1)-   SEQ ID No.: 14: Reverse primer of HumanGPR49 (Table 1)-   SEQ ID No.: 15: Forward primer of HumanNestin (Table 1)-   SEQ ID No.: 16: Reverse primer of HumanNestin (Table 1)-   SEQ ID No.: 17: Forward primer of HumanABCG2 (Table 1)-   SEQ ID No.: 18: Reverse primer of HumanABCG2 (Table 1)-   SEQ ID No.: 19: Forward primer of HumanSHH (Table 1)-   SEQ ID No.: 20: Reverse primer of HumanSHH (Table 1)-   SEQ ID No.: 21: Forward primer of HumanPtch1 (Table 1)-   SEQ ID No.: 22: Reverse primer of HumanPtch1 (Table 1)-   SEQ ID No.: 23: Forward primer of HumanSmo (Table 1)-   SEQ ID No.: 24: Reverse primer of HumanSmo (Table 1)-   SEQ ID No.: 25: Forward primer of HumanGli1 (Table 1)-   SEQ ID No.: 26: Reverse primer of HumanGli1 (Table 1)-   SEQ ID No.: 27: Forward primer of HumanGli2 (Table 1)-   SEQ ID No.: 28: Reverse primer of HumanGli2 (Table 1)-   SEQ ID No.: 29: Forward primer of Humanbeta-actin (Table 1)-   SEQ ID No.: 30: Reverse primer of Humanbeta-actin (Table 1)-   SEQ ID No.: 31: Insertion sequence of shRNA shGPR40-587 (Table 2)-   SEQ ID No.: 32: Insertion sequence of shRNA shGPR40-588 (Table 2)-   SEQ ID No.: 33: Insertion sequence of shRNA shGPR40-589 (Table 2)-   SEQ ID No.: 34: Insertion sequence of shRNA BNon-Target (NT) (Table    2)-   SEQ ID No.: 35: Gene sequence encoding R-spondin 1 (RSPO1) (OMIM:    609595; NM_001242908) (transcript variant 2)-   SEQ ID No.: 35: Gene sequence encoding R-spondin 1 (RSPO1) (OMIM:    609595; NM_001242908) (transcript variant 2)-   SEQ ID No.: 36: Amino acid sequence of R-spondin 1 (RSPO1) (OMIM:    609595; NM_001242908) (transcript variant 2)-   SEQ ID No.: 37: Gene sequence encoding R-spondin 1 (RSPO1) (OMIM:    609595; NM_001242909) (transcript variant 3)-   SEQ ID No.: 38: Amino acid sequence of R-spondin 1 (RSPO1) (OMIM:    609595; NM_001242909) (transcript variant 3)-   SEQ ID No.: 39: Gene sequence encoding R-spondin 1 (RSPO1) (OMIM:    609595; NM_001242910) (transcript variant 4)-   SEQ ID No.: 40: Amino acid sequence of R-spondin 1 (RSPO1) (OMIM:    609595; NM_001242910) (transcript variant 4)-   SEQ ID No.: 41: Gene acid sequence encoding R-spondin 4 (RSPO4)    (OMIM: 610573; NM_001040007) (transcript variant 2)-   SEQ ID No.: 42: Amino acid sequence of R-spondin 4 (RSPO4) (OMIM:    610573; NM_001040007) (transcript variant 2)-   SEQ ID No.: 43: Nucleic acid sequence of ptch1 <NM_001083602.1>-   SEQ ID No.: 44: Amino acid sequence of ptch1 <NM_001083602.1>-   SEQ ID No.: 45: Nucleic acid sequence of gli1 <NM_001167609.1>-   SEQ ID . No.: 46: Amino acid sequence of gli1 <NM_001167609.1>-   SEQ ID No.: 47: Nucleic acid sequence of gli2 <NM_005270.4>-   SEQ ID No.: 48: Amino acid sequence of gli2 <NM_005270.4>-   SEQ ID No.: 49: Nucleic acid sequence of lrp6 <NM_002336.2>-   SEQ ID No.: 50: Amino acid sequence of lrp6 <NM_002336.2>-   SEQ ID NO.: 51: Nucleic acid sequence of β-catenin <NM_131059.2>-   SEQ ID No.: 52: Amino acid sequence of β-catenin <NM_131059.2>

1)-11) (canceled) 12) A method for treating a corneal endothelial celldisorder or preventing the progression of a corneal endothelial celldisorder in a subject, comprising administering one or more R-spondinsor a functional equivalent thereof to the subject. 13)-37) (canceled)38) The method according to claim 1, wherein the R-spondins comprise oneor more R-spondins selected from R-spondin 1, R-spondin 2, R-spondin 3,R-spondin 4 and combinations thereof. 39) The method according to claim1, wherein the R-spondins comprise R-spondin 1.